JP2024056935A - Single domain antibody-containing ligand binding molecules - Google Patents

Abstract

[Problem] To provide a single domain antibody-containing ligand-binding molecule that selectively activates a ligand such as a cytokine or chemokine in a target tissue, a complex of the ligand-binding molecule and the ligand, a fusion protein containing the ligand-binding molecule, and methods for producing them. [Solution] The present invention relates to a single domain antibody-containing ligand-binding molecule whose binding activity to a ligand is reduced by cleavage of the cleavage site and a method for producing the same, a complex formed by the ligand-binding molecule and a ligand, a fusion protein containing the ligand-binding molecule and a ligand, and a pharmaceutical composition containing the ligand-binding molecule or a fusion protein of the ligand-binding molecule and a ligand. [Selected Figure] Figure 1

Description

The present invention provides a single domain antibody-containing ligand-binding molecule, a method for producing the ligand-binding molecule, and a pharmaceutical composition containing the ligand-binding molecule, as well as a fusion protein containing the ligand-binding molecule, a method for producing the fusion protein, and a pharmaceutical composition containing the fusion protein.

Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Many IgG-type antibody drugs are on the market, and many antibody drugs are currently being developed (Non-Patent Document 1 and Non-Patent Document 2).

As cancer treatment drugs using antibody drugs, Rituxan, which targets the CD20 antigen, Cetuximab, which targets the EGFR antigen, and Herceptin, which targets the HER2 antigen, have been approved (Non-Patent Document 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC, signal inhibition, etc.

In addition, a method is known in which a ligand having biological activity such as a cytokine is delivered to a solid tumor using immunocytokines, which are antibody molecules that bind to cancer antigens highly expressed in cancer cells and are fused with the ligand. The cytokines delivered to solid tumors by the immunocytokines activate the immune system and exert an antitumor effect. Since cytokines such as IL-2, IL-12, and TNF are highly toxic, it is expected that these cytokines can be delivered to the tumor site using antibodies to act locally on the tumor, thereby reducing side effects and enhancing efficacy (Non-Patent Documents 4, 5, 6). However, none of these drugs have been approved as medicines yet due to issues such as insufficient clinical efficacy when administered systemically, a narrow therapeutic window, and high toxicity that precludes systemic administration.

The main reason for this is that even if it is an immunocytokine, when it is administered systemically, it is exposed to the entire body, and therefore may act systemically and exert toxicity, or it can only be administered in extremely low doses to avoid toxicity. There is also a report that there was no difference in the antitumor effect between an immunocytokine in which IL-2 was fused to an antibody that binds to a cancer antigen and an immunocytokine in which IL-2 was fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078 The therapeutic antibody market to 2008. Pavlou AK, Belsey MJ, Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327 Cyclophosphamide and tucotuzumab (huKS-IL2) following first-line chemotherapy in responding patients with extensive-disease small-cell lung cancer. Gladkov O, Ramlau R, Serwatowski P, Milanowski J, Tomeczko J, Komarnitsky PB, Kramer D, Krzakowski MJ. Anticancer Drugs. 2015 Nov;26(10):1061-8. Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma. Paoloni M, Mazcko C, Selting K, Lana S, Barber L, Phillips J, Skorupski K, Vail D, Wilson H, Biller B, Avery A, Kiupel M, LeBlanc A, Bernhardt A, Brunkhorst B, Tighe R, Khanna C. PLoS One. 2015 Jun 19;10(6):e0129954. Isolated limb perfusion with the tumor-targeting human monoclonal antibody-cytokine fusion protein L19-TNF plus melphalan and mild hyperthermia in patients with locally advanced extremity melanoma. Papadia F, Basso V, Patuzzo R, Maurichi A, Di Florio A, Zardi L, Ventura E, Gonzalez-Iglesias R, Lovato V, Giovannoni L, Tasciotti A, Neri D, Santinami M, Menssen HD, De Cian F. J Surg Oncol. 2013 Feb;107(2):173-9. Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution. Tzeng A, Kwan BH, Opel CF, Navaratna T, Wittrup KD. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3320-5.

The present invention has been made in view of these circumstances, and one of its objects is to provide a single domain antibody-containing ligand-binding molecule that selectively activates a ligand such as a cytokine or chemokine in a target tissue, a complex of the ligand-binding molecule and the ligand, a fusion protein containing the ligand-binding molecule, and a method for producing them. Another object of the present invention is to provide a pharmaceutical composition that contains as an active ingredient the ligand-binding molecule, or the complex of the ligand-binding molecule and the ligand, or the fusion protein containing the ligand-binding molecule, and a method for producing the pharmaceutical composition.

The inventors have conducted intensive research to achieve the above object, and have created a single domain antibody-containing ligand-binding molecule containing a cleavage site and a fusion protein containing the ligand-binding molecule. The inventors have also found that the ligand-binding molecule, a complex of the ligand-binding molecule and the ligand, a fusion protein containing the ligand-binding molecule, or a pharmaceutical composition containing them are useful for treating a disease using the ligand, and have found that the ligand-binding molecule, a complex of the ligand-binding molecule and the ligand, or a fusion protein containing the ligand-binding molecule are useful for treating a disease including administering the ligand-binding molecule, or a complex of the ligand-binding molecule and the ligand, or a fusion protein containing the ligand-binding molecule, and that the ligand-binding molecule or a fusion protein containing the ligand-binding molecule is useful in the manufacture of a medicine for treating a disease. The inventors have also created a method for producing the ligand-binding molecule, or a complex of the ligand-binding molecule and the ligand, or a fusion protein containing the ligand-binding molecule, thereby completing the present invention.

The present invention is based on these findings, and specifically includes the embodiments exemplified below.
(A1) A ligand-binding molecule, comprising a single domain antibody capable of binding to a ligand and having at least one cleavage site introduced therein, such that binding of the ligand-binding molecule to the ligand when the cleavage site is cleaved is weakened compared to binding of the ligand-binding molecule to the ligand when the cleavage site is uncleaved.
(A2) The ligand-binding molecule according to (A1), wherein when the cleavage site is cleaved, the ligand is released from the ligand-binding molecule.
(A3) The ligand-binding molecule according to (A1) or (A2), wherein the cleavage site comprises a protease cleavage sequence.
(A4) The ligand-binding molecule according to (A3), wherein the protease is a target tissue-specific protease.
(A5) The ligand-binding molecule according to (A4), wherein the target tissue is a cancer tissue, and the target tissue-specific protease is a cancer tissue-specific protease.
(A6) The ligand-binding molecule according to (A4), wherein the target tissue is an inflamed tissue, and the target tissue-specific protease is an inflamed tissue-specific protease.
(A7) The ligand-binding molecule according to any one of (A3) to (A6), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloproteases.
(A8) The ligand-binding molecule according to any one of (A3) to (A7), wherein the protease cleavage sequence is a sequence including one or more sequences selected from the sequences shown in SEQ ID NOs: 2 to 82, 788 to 827, and the sequences listed in Table 1.
(A9) The ligand-binding molecule according to any one of (A3) to (A8), further comprising a first flexible linker added to one end of the protease cleavage sequence.
(A10) The ligand-binding molecule according to (A9), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer.
(A11) The ligand-binding molecule according to (A9) or (A10), further comprising a second flexible linker added to the other end of the protease cleavage sequence.
(A12) The ligand-binding molecule according to (A11), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer.
(A13) A ligand-binding molecule according to any one of (A1) to (A12), wherein the single domain antibody is a VHH, or a single domain VH antibody, or a single domain VL antibody.
(A14) The ligand-binding molecule according to (A13), wherein the single domain antibody is a VHH or a single domain VH antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and a first flexible linker, or the protease cleavage sequence, the first flexible linker and the second flexible linker are introduced into one or more positions contained in one or more sequences selected from the following sequences of the single domain antibody: Single domain antibody: sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering); single domain antibody: sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering); single domain antibody: sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering); single domain antibody: sequence from amino acid 55 (Kabat numbering) to the sequence up to amino acid number 69 (Kabat numbering), single domain antibody, sequence from amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), single domain antibody, sequence from amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 102 (Kabat numbering), single domain antibody, sequence from amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering).
(A15) The ligand-binding molecule according to (A13), wherein the single domain antibody is a single domain VL antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker and the second flexible linker are introduced into one or more positions contained in one or more sequences selected from the following sequences of the single domain antibody: Single domain antibody: sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering); single domain antibody: sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering); single domain antibody: sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering); single domain antibody: sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering); single domain antibody: sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering); single domain antibody: sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering); single domain antibody: sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering).
(A16) A ligand-binding molecule described in any one of (A1) to (A15), wherein the ligand is a molecule having biological activity, and the single domain antibody inhibits the biological activity of the ligand by binding to the ligand.
(A17) A ligand-binding molecule described in any one of (A1) to (A16), wherein the ligand is a molecule having biological activity, and the single domain antibody has neutralizing activity against the ligand.
(A18) A ligand-binding molecule according to any one of (A1) to (A17), wherein the ligand-binding molecule comprises only a single domain antibody comprising the cleavage site or protease cleavage sequence.
(A19) The ligand-binding molecule according to any one of (A1) to (A18), further comprising an antibody Fc region.
(A20) A ligand-binding molecule according to any one of (A1) to (A19), which comprises, from the N-terminus to the C-terminus, a continuous peptide chain consisting of a single domain antibody-antibody Fc region.
(A21) The ligand-binding molecule according to any one of (A1) to (A19), which is a dimer comprising two consecutive peptide chains consisting of a single domain antibody-antibody hinge region-antibody Fc region.
(A22) The ligand-binding molecule according to any one of (A19) to (A21), wherein the antibody Fc region is an Fc region comprising one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103 to 106, or an Fc region mutant obtained by modifying any of these Fc regions.
(A23) A ligand-binding molecule according to any one of (A1) to (A22), wherein the ligand is a cytokine or a chemokine.
(A24) The ligand-binding molecule according to any one of (A1) to (A23), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family.
(A25) A ligand binding molecule according to any one of (A1) to (A24), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra.
(A26) A ligand-binding molecule according to any one of (A1) to (A25), which is bound to the ligand.
(A27) A ligand-binding molecule according to any one of (A1) to (A25), which is fused to the ligand.
(A28) The ligand-binding molecule according to (A27), wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody contained in the ligand-binding molecule does not bind to any other ligand.
(A29) The ligand-binding molecule according to (A27) or (A28), wherein the ligand-binding molecule is fused to the ligand via a linker.
(A30) The ligand-binding molecule according to (A29), wherein the linker does not contain a protease cleavage sequence.
(A31) A complex formed between the ligand and the ligand-binding molecule according to any one of (A1) to (A25).
(A32) A fusion protein in which the ligand is fused to the ligand-binding molecule according to any one of (A1) to (A25).
(A33) The fusion protein described in (A32), wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody does not bind to another ligand.
(A34) The fusion protein according to (A32) or (A33), wherein the ligand-binding molecule is fused to the ligand via a linker.
(A35) The fusion protein according to (A34), wherein the linker does not contain a protease cleavage sequence.
(A36) The fusion protein according to (A34) or (A35), in which the ligand-linker-ligand-binding molecule are fused in this order from the N-terminus to the C-terminus.
(B1) A ligand-binding molecule, the ligand-binding molecule comprising a single domain antibody capable of binding to a ligand and comprising at least one protease cleavage sequence, the protease cleavage sequence being cleavable by a target tissue-specific protease, wherein binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is cleaved is weakened compared to binding of the ligand-binding molecule to the ligand when the cleavage site is uncleaved.
(B2) The ligand-binding molecule according to (B1), wherein, when the protease cleavage sequence is cleaved, the ligand is released from the ligand-binding molecule.
(B3) The ligand-binding molecule according to any one of (B1) to (B2), wherein the target tissue is a cancer tissue, and the target tissue-specific protease is a cancer tissue-specific protease.
(B4) The ligand-binding molecule according to (B1) to (B2), wherein the target tissue is an inflamed tissue, and the target tissue-specific protease is an inflamed tissue-specific protease.
(B5) The ligand-binding molecule according to any one of (B1) to (B4), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloproteases.
(B6) The ligand-binding molecule according to any one of (B1) to (B5), wherein the protease cleavage sequence is a sequence including one or more sequences selected from the sequences shown in SEQ ID NOs: 2 to 82, 788 to 827, and the sequences listed in Table 1.
(B7) A ligand-binding molecule according to any one of (B1) to (B6), further comprising a first flexible linker added to one end of the protease cleavage sequence.
(B8) The ligand-binding molecule described in (B7), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer.
(B9) The ligand-binding molecule according to (B7) or (B8), further comprising a second flexible linker added to the other end of the protease cleavage sequence.
(B10) The ligand-binding molecule according to (B9), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer.
(B11) A ligand-binding molecule according to any one of (B1) to (B10), wherein the single domain antibody is a VHH, or a single domain VH antibody, or a single domain VL antibody.
(B12) The ligand-binding molecule according to (B11), wherein the single domain antibody is a VHH or a single domain VH antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and a first flexible linker, or the protease cleavage sequence, the first flexible linker and the second flexible linker are introduced into one or more positions contained in one or more sequences of the single domain antibody selected from the following sequences: Single domain antibody: sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering); single domain antibody: sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering); single domain antibody: sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering); single domain antibody: sequence from amino acid 55 (Kabat numbering) to the sequence up to amino acid number 69 (Kabat numbering), single domain antibody, sequence from amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), single domain antibody, sequence from amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 102 (Kabat numbering), single domain antibody, sequence from amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering).
(B13) The ligand-binding molecule according to (B11), wherein the single domain antibody is a single domain VL antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker and the second flexible linker are introduced into one or more positions contained in one or more sequences selected from the following sequences of the single domain antibody: Single domain antibody: sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering); single domain antibody: sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering); single domain antibody: sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering); single domain antibody: sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering); single domain antibody: sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering); single domain antibody: sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering); single domain antibody: sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering).
(B14) A ligand-binding molecule described in any one of (B1) to (B13), wherein the ligand is a molecule having biological activity, and the single domain antibody inhibits the biological activity of the ligand by binding to the ligand.
(B15) A ligand-binding molecule according to any one of (B1) to (B14), wherein the ligand is a molecule having biological activity, and the single domain antibody has neutralizing activity against the ligand.
(B16) The ligand-binding molecule according to any one of (B1) to (B15), wherein the ligand-binding molecule comprises only a single domain antibody that contains the cleavage site or protease cleavage sequence.
(B17) The ligand-binding molecule according to any one of (B1) to (B16), further comprising an antibody Fc region.
(B18) The ligand-binding molecule according to any one of (B1) to (B17), which comprises, from the N-terminus to the C-terminus, a continuous peptide chain consisting of a single domain antibody-antibody Fc region.
(B19) The ligand-binding molecule according to any one of (B1) to (B17), which is a dimer comprising two consecutive peptide chains consisting of a single domain antibody-antibody hinge region-antibody Fc region.
(B20) The ligand-binding molecule according to any one of (B17) to (B19), wherein the antibody Fc region is an Fc region comprising one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103 to 106, or an Fc region mutant obtained by modifying any of these Fc regions.
(B21) A ligand-binding molecule according to any one of (B1) to (B20), wherein the ligand is a cytokine or a chemokine.
(B22) The ligand-binding molecule according to any one of (B1) to (B21), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family.
(B23) The ligand-binding molecule according to any one of (B1) to (B22), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra.
(B24) A ligand-binding molecule according to any one of (B1) to (B23), which is bound to the ligand.
(B25) A ligand-binding molecule according to any one of (B1) to (B23), which is fused to the ligand.
(B26) The ligand-binding molecule according to (B25), wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody contained in the ligand-binding molecule does not bind to any other ligand.
(B27) The ligand-binding molecule according to (B25) or (B26), wherein the ligand-binding molecule is fused to the ligand via a linker.
(B28) The ligand-binding molecule according to (B27), wherein the linker does not contain a protease cleavage sequence.
(B29) A complex formed between the ligand and the ligand-binding molecule according to any one of (B1) to (B23).
(B30) A fusion protein in which the ligand is fused to the ligand-binding molecule according to any one of (B1) to (B23).
(B31) The fusion protein according to (B30), wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody does not bind to another ligand.
(B32) The fusion protein according to (B30) or (B31), wherein the ligand-binding molecule is fused to the ligand via a linker.
(B33) The fusion protein according to (B32), wherein the linker does not contain a protease cleavage sequence.
(B34) The fusion protein according to (B32) or (B33), in which the ligand-linker-ligand-binding molecule are fused in this order from the N-terminus to the C-terminus.
(C1) A ligand-binding molecule comprising a single domain antibody, the single domain antibody being a VHH or a single domain VH antibody, the single domain antibody having a cleavage site at one or more positions contained in one or more sequences selected from the following sequences: a sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering) of a single domain antibody, a sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering) of a single domain antibody, a sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering) of a single domain antibody, a sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering) of a single domain antibody, a sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering) of a single domain antibody, a sequence from amino acid 55 (Kabat numbering) to the sequence up to amino acid number 69 (Kabat numbering), single domain antibody, sequence from amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), single domain antibody, sequence from amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 102 (Kabat numbering), single domain antibody, sequence from amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering).
(C2) A ligand-binding molecule comprising a single domain antibody, the single domain antibody being a single domain VL antibody, the single domain antibody having a cleavage site at one or more positions contained in one or more sequences selected from the following sequences: Single domain antibody: sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering); single domain antibody: sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering); single domain antibody: sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering); single domain antibody: sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering); single domain antibody: sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering); single domain antibody: sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering); single domain antibody: sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering).
(C3) The ligand-binding molecule described in (C1) or (C2), wherein the single domain antibody is capable of binding to the ligand, and the binding of the ligand-binding molecule to the ligand when the cleavage site is cleaved is weakened compared to the binding of the ligand-binding molecule to the ligand when the cleavage site is uncleaved.
(C4) The ligand-binding molecule according to any one of (C1) to (C3), wherein when the cleavage site is cleaved, the ligand is released from the ligand-binding molecule.
(C5) A ligand binding molecule according to any one of (C1) to (C4), wherein the cleavage site comprises a protease cleavage sequence.
(C6) The ligand-binding molecule according to (C5), wherein the protease is a target tissue-specific protease.
(C7) The ligand-binding molecule according to (C6), wherein the target tissue is a cancer tissue, and the target tissue-specific protease is a cancer tissue-specific protease.
(C8) The ligand binding molecule according to (C6), wherein the target tissue is an inflamed tissue and the target tissue-specific protease is an inflamed tissue-specific protease.
(C9) The ligand-binding molecule according to any one of (C5) to (C8), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloproteases.
(C10) The ligand-binding molecule according to any one of (C5) to (C9), wherein the protease cleavage sequence is a sequence including one or more sequences selected from the sequences shown in SEQ ID NOs: 2 to 82, 788 to 827, and the sequences listed in Table 1.
(C11) The ligand-binding molecule according to any one of (C5) to (C10), further comprising a first flexible linker added to one end of the protease cleavage sequence.
(C12) The ligand-binding molecule according to (C11), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer.
(C13) The ligand-binding molecule according to (C11) or (C12), further comprising a second flexible linker added to the other end of the protease cleavage sequence.
(C14) The ligand-binding molecule according to (C13), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer.
(C15) A ligand-binding molecule described in any one of (C1) to (C14), wherein the ligand is a molecule having biological activity, and the single domain antibody inhibits the biological activity of the ligand by binding to the ligand.
(C16) A ligand-binding molecule according to any one of (C1) to (C15), wherein the ligand is a molecule having biological activity, and the single domain antibody has neutralizing activity against the ligand.
(C17) The ligand-binding molecule according to any one of (C1) to (C16), wherein the ligand-binding molecule comprises only a single domain that includes the cleavage site or protease cleavage sequence.
(C18) The ligand-binding molecule according to any one of (C1) to (C17), wherein the ligand-binding molecule further comprises an antibody Fc region.
(C19) A ligand-binding molecule according to any one of (C1) to (C18), wherein the ligand-binding molecule comprises, from the N-terminus to the C-terminus, a continuous peptide chain consisting of a single domain antibody-antibody Fc region.
(C20) The ligand-binding molecule according to any one of (C1) to (C18), wherein the ligand-binding molecule is a dimer comprising two consecutive peptide chains consisting of a single domain antibody-antibody hinge region-antibody Fc region.
(C21) The ligand-binding molecule according to any one of (C18) to (C20), wherein the antibody Fc region is an Fc region comprising one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103 to 106, or an Fc region mutant obtained by modifying any of these Fc regions.
(C22) A ligand binding molecule according to any one of (C1) to (C21), wherein the ligand is a cytokine or a chemokine.
(C23) The ligand-binding molecule according to any one of (C1) to (C22), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family.
(C24) The ligand binding molecule according to any one of (C1) to (C23), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra.
(C25) A ligand-binding molecule according to any one of (C1) to (C24), which is bound to the ligand.
(C26) A ligand-binding molecule according to any one of (C1) to (C24), which is fused to the ligand.
(C27) The ligand-binding molecule according to (C26), wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody contained in the ligand-binding molecule does not bind to another ligand.
(C28) The ligand-binding molecule according to (C26) or (C27), wherein the ligand-binding molecule is fused to the ligand via a linker.
(C29) The ligand-binding molecule according to (C28), wherein the linker does not contain a protease cleavage sequence.
(C30) A complex formed between the ligand and the ligand-binding molecule according to any one of (C1) to (C24).
(C31) A fusion protein in which the ligand is fused to the ligand-binding molecule according to any one of (C1) to (C24).
(C32) The fusion protein according to (C31), wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody does not bind to another ligand.
(C33) The fusion protein according to (C31) or (C32), wherein the ligand-binding molecule is fused to the ligand via a linker.
(C34) The fusion protein according to (C33), wherein the linker does not contain a protease cleavage sequence.
(C35) The fusion protein according to (C33) or (C34), wherein the ligand-linker-ligand binding molecule are fused in this order from the N-terminus to the C-terminus.
(D1) A pharmaceutical composition comprising a ligand-binding molecule according to any one of (A1) to (A27), (B1) to (B25), and (C1) to (C26).
(D2) A pharmaceutical composition comprising a ligand-binding molecule according to any one of (A1) to (A26), (B1) to (B24), and (C1) to (C25) and a ligand.
(D3) A pharmaceutical composition comprising the conjugate according to (A31), (B29) or (C30).
(D4) A pharmaceutical composition comprising the fusion protein according to any one of (A32) to (A36), (B30) to (B34), and (C31) to (C35).
(E1) A method for producing a ligand-binding molecule according to any one of (A1) to (A25), (B1) to (B24), and (C1) to (C25).
(E2) The method according to (E1), comprising introducing a protease cleavage sequence into a single domain antibody in a ligand-binding molecule comprising a single domain antibody.
(E3) A method for producing a fusion protein described in any one of (A32) to (A36), (B30) to (B34), and (C31) to (C35), comprising fusing a single domain antibody-containing ligand-binding molecule having a protease cleavage sequence introduced therein with a ligand capable of binding to the single domain antibody.
(E4) A polynucleotide encoding a ligand-binding molecule according to any one of (A1) to (A25), (B1) to (B23), and (C1) to (C24).
(E5) A vector comprising the polynucleotide according to (E4).
(E6) A host cell comprising the polynucleotide according to (E4) or the vector according to (E5).
(E7) A method for producing a ligand-binding molecule according to any one of (A1) to (A25), (B1) to (B23), and (C1) to (C24), comprising the step of culturing a host cell according to (E6).
(E8) A polynucleotide encoding a fusion protein according to any one of (A32) to (A36), (B30) to (B34), and (C31) to (C35).
(E9) A vector comprising the polynucleotide according to (E8).
(E10) A host cell comprising the polynucleotide according to (E8) or the vector according to (E9).
(E11) A method for producing a fusion protein described in any one of (A32) to (A36), (B30) to (B34), and (C31) to (C35), comprising a step of culturing the host cell described in (E10).

1 is a diagram showing an example of a ligand-binding molecule of the present invention, in which when the cleavage site contained in the single domain antibody is in an uncleaved state, the single domain antibody can bind to a ligand, and when the cleavage site is in a cleaved state, the single domain antibody is cleaved and cannot bind to the ligand, resulting in the release of the ligand. 1 shows an example of a fusion protein of a ligand-binding molecule and a ligand of the present invention, in which when the cleavage site contained in the single domain antibody is not cleaved, the single domain antibody in the fusion protein can bind to the ligand in the fusion protein, and when the cleavage site is cleaved, the single domain antibody is cleaved and cannot bind to the ligand, and a part of the fusion protein containing the ligand is released. 1 shows another example of a ligand-binding molecule of the present invention, in which the ligand-binding molecule is a dimeric protein comprising a single domain antibody-antibody hinge region-antibody Fc region. 1 shows another example of a fusion protein of a ligand-binding molecule and a ligand of the present invention, in which the ligand-binding molecule is a dimeric protein comprising a single domain antibody-antibody hinge region-antibody Fc region. 1 shows the results of SDS-PAGE of protease-treated and untreated ligand-binding molecules. While the bands of the control molecules not containing a protease cleavage sequence (lanes 12 and 13) were at the same position regardless of whether they were treated with or untreated with a protease, the ligand-binding molecules into which each protease cleavage sequence had been introduced had new bands that appeared only after protease treatment, indicating that the single-domain antibody-containing ligand-binding molecules into which each protease cleavage sequence had been introduced were cleaved by protease treatment. These are real-time binding graphs evaluating the binding of protease-treated and protease-untreated ligand-binding molecules to IL-6R. The title of each figure is the name of the measured sample, the vertical axis shows the relative binding of the ligand-binding molecule to IL-6R, and the horizontal axis shows time (s). The gray line shows the data for the protease-untreated sample, and the black line shows the data for the protease-treated sample. 1 shows the results of SDS-PAGE of protease-treated and non-protease-treated fusion proteins. While the bands of the control molecule not containing a protease cleavage sequence were at the same position regardless of whether it was treated with or not with a protease, each fusion protein containing a single domain antibody-containing ligand-binding molecule into which a protease cleavage sequence had been introduced had a new band that appeared only after protease treatment, indicating that each fusion protein containing a single domain antibody-containing ligand-binding molecule into which each protease cleavage sequence had been introduced was cleaved by protease treatment. This figure shows the continuation of Figure 7-1. This figure shows the continuation of Figure 7-2. This figure shows the continuation of Figure 7-3. This is a real-time graph evaluating the binding of free IL-6R to IL6R90-bio in solutions of protease-treated and protease-untreated fusion proteins. The title of each figure is the name of the measurement sample, the vertical axis shows the relative binding of IL-6R to the biotinylated anti-IL-6R single domain antibody-containing molecule (IL6R90-bio), and the horizontal axis shows time (s). The gray line shows the data of the protease-untreated sample, and the black line shows the data of the protease-treated sample. This figure shows the continuation of Figure 8-1. FIG. 1 shows the results of SDS-PAGE of a fusion protein treated with a protease and a fusion protein not treated with a protease. Figure 1 shows real-time graphs of binding of free human PD-1 to biotinylated anti-PD-1 single domain antibody-containing molecules (PD1-bio) in solutions of protease-treated and protease-untreated fusion proteins. The title of each figure is the name of the measured sample, the vertical axis shows the relative binding of PD-1 to PD1-bio, and the horizontal axis shows time (s). The gray line shows the data for the protease-untreated sample, and the black line shows the data for the protease-treated sample. FIG. 1 shows the results of protease cleavage of IgG into which various protease cleavage sequences have been introduced. FIG. 1 shows the results of protease cleavage of IgG into which various protease cleavage sequences have been introduced. FIG. 1 shows the results of protease cleavage of IgG into which various protease cleavage sequences have been introduced.

Polypeptide : The polypeptide of the present invention generally refers to a peptide or protein having a length of about 4 amino acids or more. When a series of amino acids linked by peptide bonds from the N-terminus to the C-terminus is considered to be a peptide chain, the polypeptide of the present invention may be a complex protein formed by interactions such as SS bonds, hydrophobic interactions, and ionic bonds between a plurality of series of peptide chains. In addition, the polypeptide of the present invention is generally a polypeptide consisting of an artificially designed sequence, but is not particularly limited, and may be, for example, a polypeptide derived from an organism. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Furthermore, fragments of the above polypeptides are also included in the polypeptide of the present invention.

Amino acids . As used herein, amino acids are represented by one-letter or three-letter codes, or both, e.g., Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F, Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G, Trp/W, His/H, Tyr/Y, Ile/I, Val/V.

For the modification of amino acids in the amino acid sequence of an amino acid- modified polypeptide, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. In addition, as a method for modifying amino acids by substituting amino acids other than natural amino acids, several known methods can also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, can be suitably used.

As used herein, the meaning of the term "and/or" used to describe the site of amino acid modification includes any combination of "and" and "or." Specifically, for example, "amino acids 37, 45, and/or 47 are substituted" includes the following amino acid modification variations:
(a) No. 37, (b) No. 45, (c) No. 47, (d) No. 37 and No. 45, (e) No. 37 and No. 47, (f) No. 45 and No. 47, (g) No. 37, No. 45 and No. 47.

In the present specification, as an expression for an amino acid modification, an expression in which the one-letter code or three-letter code of the amino acid before and after the modification is written before and after a number indicating a specific position may be appropriately used. For example, the modification F37V or Phe37Val used when making a substitution of an amino acid contained in an antibody variable region represents a substitution of Phe at position 37 represented by Kabat numbering with Val. That is, the number represents the position of the amino acid represented by Kabat numbering, the one-letter code or three-letter code of the amino acid written before it represents the amino acid before the substitution, and the one-letter code or three-letter code of the amino acid written after it represents the amino acid after the substitution. Similarly, the modification P238A or Pro238Ala used when making a substitution of an amino acid in the Fc region contained in an antibody constant region represents a substitution of Pro at position 238 represented by EU numbering with Ala. That is, the number represents the position of the amino acid represented by EU numbering, the one-letter code or three-letter code of the amino acid written before it represents the amino acid before the substitution, and the one-letter code or three-letter code of the amino acid written after it represents the amino acid after the substitution.

Antibodies and Antibody Fragments The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

The term "variable region " or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of an antibody's heavy and light chains (VH and VL, respectively) usually have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen-binding specificity.

CDR
The term "complementarity determining region" or "CDR" as used herein refers to each region of an antibody variable domain or single domain antibody which is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops") and/or antigen contact residues ("antigen contacts").
Typically, antibodies contain six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary antibody CDRs herein include the following:
(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) the CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
(d) A combination of (a), (b), and/or (c), comprising CDR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), or 94-102 (H3).

Typically, a single domain antibody comprises three CDRs: CDR1, CDR2, CDR3. When the single domain antibody is a VHH or a single domain VH antibody, the CDRs of the single domain antibody illustratively comprise:
(a) hypervariable loops occurring at amino acid residues 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) the CDRs occurring at amino acid residues 31-35b (CDR1), 50-65 (CDR2), and 95-102 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts that occur at amino acid residues 30-35b (CDR1), 47-58 (CDR2), and 93-101 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
(d) A combination of (a), (b), and/or (c), comprising CDR amino acid residues 26-35 (CDR1), 26-35b (CDR1), 49-65 (CDR2), 93-102 (CDR3), or 94-102 (CDR3).

When the single domain antibody is a single domain VL antibody, the CDRs of the single domain antibody illustratively include:
(a) hypervariable loops occurring at amino acid residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) the CDRs occurring at amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts that occur at amino acid residues 27c-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
(d) a combination of (a), (b), and/or (c), comprising CDR amino acid residues 46-56 (CDR2), 47-56 (CDR2), 48-56 (CDR2), or 49-56 (CDR2).

Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

FR
"Framework" or "FR" refers to the variable domain or single domain antibody residues other than the complementarity determining region (CDR) residues. The FR of a variable domain or single domain antibody usually consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the CDRs and FRs usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In single domain antibodies, the sequences of the CDRs and FRs usually appear in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al., supra.

Constant Region The term "constant region" or "constant domain" as used herein refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded, with each heavy chain having, from the N-terminus to the C-terminus, a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by a heavy chain constant region (CH) that includes a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of a native antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. The term "comprising a constant region" may include the entire constant region or a portion of the constant region.

The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

Fc Region The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, for human IgG1, the heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain, with the exception that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

Hinge Region The term "hinge region" or "antibody hinge region" can refer to a region consisting of amino acids 216 to 230 (EU numbering) in an antibody heavy chain, or a portion thereof.

Single domain antibody: As used herein, the term "single domain antibody" refers to an antibody that can exert antigen-binding activity by its domain alone. The structure of a single domain antibody is not limited as long as the domain alone can exert antigen-binding activity. Conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing VH and VL, whereas single domain antibodies are known to be able to exert antigen-binding activity by the domain structure of the single domain antibody alone, without pairing with other domains. Single domain antibodies usually have a relatively low molecular weight and exist in the form of a monomer. In some embodiments, the form of a single domain antibody is similar to that of an antibody heavy chain variable region or an antibody light chain variable region.
Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules that congenitally lack light chains, such as the variable region (VHH) of camelid heavy chain antibodies and shark _VNAR , or antibody fragments that contain all or a part of the VH domain or all or a part of the VL domain of an antibody. Examples of single-domain antibodies, which are antibody fragments that contain all or a part of the VH/VL domains of an antibody, include, but are not limited to, single-domain antibodies (hereinafter referred to as single-domain VH antibodies and single-domain VL antibodies) artificially produced starting from human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516 B1, etc.

Single domain antibodies can be obtained from animals capable of producing single domain antibodies or by immunizing animals capable of producing single domain antibodies. Examples of animals capable of producing single domain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing single domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015/143414 and US Patent Publication US2011/0123527 A1. Humanized single domain antibodies can also be obtained by making the framework sequence of a single domain antibody obtained from an animal a human germline sequence or a sequence similar thereto. Humanized single domain antibodies (e.g., humanized VHH) are also an embodiment of the single domain antibody of the present invention.

Single domain antibodies can be obtained from animals capable of producing single domain antibodies or by immunizing animals capable of producing single domain antibodies. Examples of animals capable of producing single domain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing single domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015/143414 and US Patent Publication US2011/0123527 A1. Humanized single domain antibodies can also be obtained by making the framework sequence of a single domain antibody obtained from an animal a human germline sequence or a sequence similar thereto. Humanized single domain antibodies (e.g., humanized VHH) are also an embodiment of the single domain antibody of the present invention.

Single domain antibodies can also be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, etc. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11 (1691-1701)).

In the present invention, there are embodiments in which a cleavage site/protease cleavage sequence is introduced into a single domain antibody, but regardless of whether a cleavage site/protease cleavage sequence is introduced, the antibody can be referred to as a "single domain antibody."

Single domain antibodies of the invention, in some embodiments, generally comprise:
a) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 11 according to the Kabat numbering is selected from the group consisting of L, M, S, V, W, preferably L; and/or b) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 37 according to the Kabat numbering is selected from the group consisting of F, Y, H, I, L, V, preferably F or Y; and/or c) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 44 according to the Kabat numbering is selected from the group consisting of G, E, A, D, Q, R, S, L, preferably G, E or Q, more preferably G or E; and/or d) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 45 according to the Kabat numbering is selected from the group consisting of L, R, C, I, L, P, Q, V, preferably L or R; and/or e) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 47 according to the Kabat numbering is selected from the group consisting of W, L, F, A, G, I, M, R, S, V, Y, preferably W, L, F or R; and/or f) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 83 according to the Kabat numbering is selected from the group consisting of R, K, N, E, G, I, M, Q, T, preferably K or R, more preferably K; and/or g) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 84 according to the Kabat numbering is selected from the group consisting of P, A, L, R, S, T, D, V, preferably P; and/or h) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 103 according to the Kabat numbering is selected from the group consisting of W, P, R, S, preferably W; and/or i) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 104 according to the Kabat numbering is G or D, preferably G; and/or j) an amino acid sequence consisting of four framework regions/sequences interrupted by three complementarity determining regions/sequences, wherein the amino acid residue at position 108 according to the Kabat numbering is selected from the group consisting of Q, L, R, preferably Q or L.

More specifically, but not exclusively, a single domain antibody can be defined as a polypeptide comprising any one of the following amino acid sequences consisting of four framework regions/sequences interspersed with three complementarity determining regions/sequences:
k) an amino acid sequence in which the amino acid residues at positions 43 to 46 according to the Kabat numbering are KERE or KQRE;
l) an amino acid sequence in which the amino acid residues 44 to 47 according to the Kabat numbering are GLEW;
m) An amino acid sequence in which the amino acid residues at positions 83 and 84 according to the Kabat numbering are KP or EP.

Chimeric AntibodiesThe term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.A "chimeric single domain antibody" refers to a single domain antibody in which a portion of the single domain antibody is derived from a particular source or species, while the remainder of the single domain antibody is derived from a different source or species.

Humanized antibody A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In one embodiment, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all CDRs correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A "humanized single-domain antibody" refers to a chimeric single-domain antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In one embodiment, a humanized single-domain antibody contains all or substantially all CDRs corresponding to those of a non-human antibody, and all or substantially all FRs corresponding to those of a human antibody. In a humanized antibody, even if some of the residues in the FRs do not correspond to those of a human antibody, it is considered as an example in which substantially all FRs correspond to those of a human antibody. For example, when VHH, which is one aspect of a single domain antibody, is humanized, some of the residues in the FR must be changed to residues that do not correspond to those in human antibodies (C Vincke et al., The Journal of Biological Chemistry 284, 3273-3284).
A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

Affinity "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "antibody that binds to the same epitope " as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

Isolated antibody An "isolated" antibody is one that has been separated from the components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

Monoclonal Antibodies As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and/or bind to the same epitope, except for possible variants (e.g., variants including naturally occurring variants or variants that arise during the manufacture of a monoclonal antibody preparation, which are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such and other exemplary methods for making monoclonal antibodies are described herein.

"Percent (%) amino acid sequence identity" to a sequence identity reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

Vector As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. A vector can effect expression of a nucleic acid to which it is operatively linked. Such vectors are also referred to herein as "expression vectors."

Host Cells, etc. The terms "host cell,""host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

Protease As used herein, the term "protease" refers to an enzyme, such as an endopeptidase or an exopeptidase, that hydrolyzes peptide bonds, typically an endopeptidase. The protease used in the present invention is limited only by its ability to cleave the protease cleavage sequence, and the type is not particularly limited. In some embodiments, a target tissue-specific protease is used. The target tissue-specific protease is, for example,
(1) a protease that is expressed at a higher level in a target tissue than in normal tissue;
(2) a protease that has higher activity in target tissue than in normal tissue;
(3) a protease that is expressed at a higher level in target cells than in normal cells;
(4) a protease that has higher activity in target cells than in normal cells;
In a more specific embodiment, a cancer tissue-specific protease or an inflamed tissue-specific protease is used.

Target Tissue: As used herein, the term "target tissue" refers to a tissue that contains at least one target cell. In some embodiments of the invention, the target tissue is cancerous tissue. In some embodiments of the invention, the target tissue is inflamed tissue.

The term "cancer tissue" refers to tissue that contains at least one cancer cell. Thus, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such that cancer tissue contains cancer cells and blood vessels. As used herein, tumor mass refers to a foci of tumor tissue. The term "tumor" is used generally to refer to benign or malignant neoplasms.

As used herein, "inflamed tissue" includes, for example, the following:
・Joints in rheumatoid arthritis and osteoarthritis ・Lungs (alveoli) in bronchial asthma and COPD
・Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis ・Fibrotic tissues in fibrosis of the liver, kidneys, and lungs ・Tissues undergoing rejection in organ transplants ・Blood vessels and the heart (myocardium) in arteriosclerosis and heart failure
・Visceral fat in metabolic syndrome ・Skin tissue in atopic dermatitis and other skin irritations ・Spinal nerves in herniated discs and chronic lower back pain

Target tissue-specific proteases are known that are specifically expressed or specifically activated in several types of target tissues, or that are thought to be associated with disease states of target tissues (target tissue-specific proteases). For example, International Publication WO2013/128194, International Publication WO2010/081173, International Publication WO2009/025846, etc. disclose proteases that are specifically expressed in cancer tissues. In addition, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul; 6(7): 541-50., Nat Rev Drug Discov. 2014 Dec; 13(12): 904-27., Respir Res. 2016 Mar 4; 17: 23., Dis Model Mech. 2014 Feb; 7(2): 193-203., and Biochim Biophys Acta. 2012 Jan; 1824(1): 133-45.

In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active. In many tissues, substances that inhibit active proteases exist, and their activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, active proteases may escape inhibition and become specifically activated.
Active proteases can be measured using an antibody that recognizes active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or a method in which a peptide recognized by a protease is fluorescently labeled and quenched before cleavage but emits light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038/nrd3053.).

Specific proteases include, but are not limited to, cysteine proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (including matriptase (MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptophan, etc.), and erythrocyte proteases (including erythrocyte endothelial cell line (ECC)). metalloproteases (including membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23 and MMP26-28) metalloproteases (MMP1-28)), proteases A disintegrin and metalloproteases (ADAMs), metalloproteases with A disintegrin or thrombospondin motifs (ADAMTSs), meprins (meprin α alpha), meprin beta), CD10 (CALLA), as well as prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein alpha (FAP), granzyme B, guanidinobenzoatase (GB), hepsin, neprilysin, NS3/4A, HCV-NS3/4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V/L2, cathepsin X/Z/P, cruzipain, otubain 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa), HtrA1, lactoferrin, marapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-Like These include proteases, AMSH, gamma secretase, antiplasmin cleaving enzyme (APCE), decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

From another perspective, a target tissue-specific protease can refer to a cancer tissue-specific protease or an inflamed tissue-specific protease.

Cancer tissue-specific proteases include proteases that are specifically expressed in cancer tissues, such as those disclosed in International Publication WO2013/128194, International Publication WO2010/081173, and International Publication WO2009/025846.

The type of cancer tissue-specific protease has a higher specificity of expression in the cancer tissue of the treatment target, and the more side effect reduction effect can be obtained.The concentration of the cancer tissue-specific protease in the cancer tissue is preferably 5 times or more higher than the concentration in the normal tissue, more preferably 10 times or more higher, even more preferably 100 times or more higher, particularly preferably 500 times or more higher, and most preferably 1000 times or more higher.In addition, the activity of the cancer tissue-specific protease in the cancer tissue is preferably 2 times or more higher than the activity in the normal tissue, more preferably 3 times or more higher, 4 times or more higher, 5 times or more higher, more preferably 10 times or more higher, even more preferably 100 times or more higher, particularly preferably 500 times or more higher, and most preferably 1000 times or more higher.
In addition, the cancer tissue-specific protease may be one that is bound to the cell membrane of the cancer cell, or one that is not bound to the cell membrane and is secreted outside the cell. When the cancer tissue-specific protease is not bound to the cell membrane of the cancer cell, in order for the cytotoxicity by the immune cell to be specific to the cancer cell, it is preferable that the cancer tissue-specific protease is present inside or near the cancer tissue. In this specification, "near the cancer tissue" means within a range in which the cancer tissue-specific protease cleavage sequence is cleaved to exert the effect of reducing the ligand binding activity. However, it is preferable that the range is one in which normal cells are not damaged as much as possible.
From another perspective, cancer tissue-specific proteases are
(i) a protease that is expressed at a higher level in cancer tissue than in normal tissue;
(ii) a protease that has higher activity in cancer tissue than in normal tissue;
(iii) proteases that are expressed at higher levels in cancer cells than in normal cells;
(iv) proteases that have higher activity in cancer cells than in normal cells;
Either:
The cancer tissue-specific protease may be one type alone or two or more types in combination. The number of types of cancer tissue-specific proteases can be appropriately determined by those skilled in the art in consideration of the type of cancer to be treated.

From the above viewpoints, among the above-mentioned proteases, preferred cancer tissue-specific proteases are serine proteases and metalloproteases, more preferred are matriptase (including MT-SP1), urokinase (uPA) and metalloproteases, and even more preferred are MT-SP1, uPA, MMP-2 and MMP-9.

Inflammatory tissue-specific protease The more specific the type of inflammatory tissue-specific protease is expressed in the inflammatory tissue of the treatment target, the more effective it is in reducing side effects. The concentration of the inflammatory tissue-specific protease in the inflammatory tissue is preferably 5 times higher than that in the normal tissue, more preferably 10 times higher, even more preferably 100 times higher, particularly preferably 500 times higher, and most preferably 1000 times higher. In addition, the activity of the inflammatory tissue-specific protease in the inflammatory tissue is preferably 2 times higher than that in the normal tissue, more preferably 3 times higher, 4 times higher, 5 times higher, or 10 times higher, more preferably 100 times higher, particularly preferably 500 times higher, and most preferably 1000 times higher.
In addition, the inflammatory tissue-specific protease may be one that is bound to the cell membrane of an inflammatory cell, or one that is not bound to the cell membrane and is secreted outside the cell. When the inflammatory tissue-specific protease is not bound to the cell membrane of an inflammatory cell, it is preferable that the inflammatory tissue-specific protease is present inside or near the inflammatory tissue, so that the cytotoxicity by the immune cell is specific to the inflammatory cell. In this specification, "near the inflammatory tissue" means within a range in which the inflammatory tissue-specific protease cleavage sequence is cleaved to exert the effect of reducing the ligand binding activity. However, it is preferable that the range is one in which normal cells are not damaged as much as possible.
From another perspective, inflammatory tissue-specific proteases are
(i) a protease that is expressed at a higher level in inflamed tissue than in normal tissue;
(ii) proteases that have higher activity in inflamed tissues than in normal tissues;
(iii) proteases that are expressed at higher levels in inflammatory cells than in normal cells;
(iv) proteases that have higher activity in inflammatory cells than in normal cells;
Either:
The inflammatory tissue-specific protease may be one type alone or two or more types in combination. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the condition of the subject to be treated.

From the above viewpoints, as the inflammatory tissue-specific protease, among the above-listed proteases, metalloproteases are preferred, and among the metalloproteases, ADAMTS5, MMP-2, MMP-7, MMP-9, and MMP-13 are more preferred.

Methods for Producing Antibodies Methods for producing antibodies having the desired binding activity are known to those skilled in the art. Below, methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples. When producing single domain antibodies, they can be produced appropriately according to the following examples, although there are differences in the animals immunized.

Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known means. As anti-IL-6R antibodies, monoclonal antibodies derived from mammals can be preferably produced. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques. Antibodies referred to in this application include "humanized antibodies" and "chimeric antibodies."

Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows. That is, a mammal is immunized according to a conventional immunization method using IL-6R protein as a sensitizing antigen. The resulting immune cells are fused with a known parent cell by a conventional cell fusion method. Next, a hybridoma that produces an anti-IL-6R antibody can be selected by screening for monoclonal antibody-producing cells using a conventional screening method.

Specifically, monoclonal antibodies are produced, for example, as follows. First, the IL-6R gene is expressed to obtain the IL-6R protein used as a sensitizing antigen for obtaining the antibody. That is, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or the culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968) is expressed. Purified natural IL-6R protein can also be used as a sensitizing antigen.

The purified IL-6R protein can be used as a sensitizing antigen for immunizing mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. It can also be obtained by decomposing the IL-6R protein using a protease, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. The number of amino acids constituting the peptide to be used as a sensitizing antigen is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

In addition, a fusion protein in which a desired partial polypeptide or peptide of the IL-6R protein is fused with a different polypeptide can be used as a sensitizing antigen. For example, an Fc fragment of an antibody or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. A method for producing a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). A method for obtaining IL-6R to be used as a sensitizing antigen and a method for immunization using the same are also specifically described in WO2003/000883, WO2004/022754, WO2006/006693, etc.

The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. In general, rodents such as mice, rats, hamsters, rabbits, monkeys, etc. are preferably used. When obtaining a single domain antibody, a camelid or a transgenic animal into which a gene capable of producing a single domain antibody has been introduced is preferably used.

The above-mentioned animals are immunized with the sensitizing antigen according to a known method. For example, as a general method, immunization is performed by administering the sensitizing antigen intraperitoneally or subcutaneously to the mammal. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, etc., is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days. In addition, a suitable carrier can be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the body of the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages:
- Immunostimulation can be achieved by maintaining the structure of membrane proteins such as IL-6R - No need to purify the immunizing antigen

To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing IL-6R protein is administered to the animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be suitably used. A commonly used method can be used as a method for administering a vector to a living body. For example, DNA immunization can be performed by introducing gold particles to which an expression vector is adsorbed into the cells of an individual animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003/104453.

After the mammal is immunized in this manner and an increase in the titer of antibodies that bind to IL-6R in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. As preferred immune cells, spleen cells in particular can be used.

Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably have an appropriate selection marker for screening. A selection marker refers to a trait that allows (or does not allow) a cell to survive under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die, but when fused with normal cells, they can continue to synthesize DNA by utilizing the salvage circuitry of normal cells, and therefore grow even in HAT selection medium.

Cells deficient in HGPRT or TK can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes that cannot incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2/0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), and S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be preferably used.

Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).
More specifically, the cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

The ratio of immune cells to myeloma cells can be set as desired. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, or other normal culture medium used for this type of cell culture, which is suitable for the growth of the myeloma cell line, can be used, and serum supplements such as fetal calf serum (FCS) can be suitably added.

For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., average molecular weight of about 1000 to 6000) that has been pre-warmed to about 37°C is added, usually at a concentration of 30 to 60% (w/v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, the appropriate culture medium listed above is successively added, and the process of centrifuging and removing the supernatant is repeated, allowing cell fusion agents and the like that are undesirable for hybridoma growth to be removed.

The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture can be continued in the above HAT culture medium for a sufficient time (usually several days to several weeks) for cells other than the desired hybridoma (non-fused cells) to die. Then, screening and single cloning of hybridomas producing the desired antibody are performed by a conventional limiting dilution method.

The hybridomas thus obtained can be selected by using a selective culture medium corresponding to the selection marker possessed by the myeloma used in the cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used in cell fusion, cells that have successfully fused with normal cells can selectively grow in the HAT culture medium. The culture is continued using the above HAT culture medium for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Next, screening and single cloning of hybridomas that produce the desired antibody can be performed by the usual limiting dilution method.

Screening and single cloning of the desired antibody can be suitably carried out by a screening method based on a known antigen-antibody reaction. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such a monoclonal antibody can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows the binding of an antibody to a cell surface to be measured by analyzing cells that have been contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

To screen for hybridomas that produce the monoclonal antibody of the present invention by FACS, first, cells expressing IL-6R are prepared. Preferred cells for screening are mammalian cells in which IL-6R is forcibly expressed. By using non-transformed mammalian cells used as host cells as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to cells that forcibly express IL-6R, hybridomas that produce IL-6R monoclonal antibodies can be obtained.

Alternatively, the binding activity of the antibody to immobilized IL-6R-expressing cells can be evaluated based on the principle of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody that has the ability to bind to the antigen and are selected by these screening methods can be cloned by limiting dilution or the like.

The hybridomas producing the monoclonal antibodies thus produced can be subcultured in a normal culture medium. The hybridomas can also be stored for long periods in liquid nitrogen.

The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be obtained from the ascites. The former method is suitable for obtaining highly pure antibodies.

Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be used suitably. The cloned antibody gene is incorporated into an appropriate vector and introduced into a host, whereby the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from a hybridoma cell producing the anti-IL-6R antibody. To do this, generally, total RNA is first extracted from the hybridoma. The following method can be used to extract mRNA from cells.
- Guanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294-5299)
-AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or the like. Alternatively, kits for extracting total mRNA directly from cells, such as QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are also commercially available. Using such a kit, mRNA can be obtained from a hybridoma. cDNA encoding an antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like. In addition, for the synthesis and amplification of cDNA, a SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be appropriately used. Furthermore, during the synthesis of such cDNA, appropriate restriction enzyme sites can be introduced at both ends of the cDNA, as described below.

The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. A recombinant vector is thus produced, introduced into E. coli or the like, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector contains the base sequence of the desired cDNA is confirmed by a known method, such as the dideoxynucleotide chain termination method.

To obtain genes encoding variable regions, it is easy to use the 5'-RACE method using primers for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template to obtain a 5'-RACE cDNA library. A commercially available kit such as the SMART RACE cDNA Amplification Kit can be used to synthesize the 5'-RACE cDNA library.

Using the obtained 5'-RACE cDNA library as a template, antibody genes are amplified by PCR. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

Specifically, for example, when the aim is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains and κ and λ chains as light chains can be used. To amplify the variable region genes of IgG, a primer that anneals to a portion corresponding to the constant region close to the variable region is generally used as the 3' primer. Meanwhile, a primer included in the 5' RACE cDNA library construction kit is used as the 5' primer.

Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The desired antibody can be screened using the binding activity of the reconstituted immunoglobulin to IL-6R as an index. For example, when the aim is to obtain an antibody against IL-6R, it is more preferable that the binding of the antibody to IL-6R is specific. Antibodies that bind to IL-6R can be screened, for example, as follows;
(1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell;
(2) detecting the binding of the antibody to IL-6R-expressing cells; and (3) selecting an antibody that binds to IL-6R-expressing cells.

Methods for detecting the binding of an antibody (including a single domain antibody) to an IL-6R-expressing cell are known. Specifically, the binding of an antibody to an IL-6R-expressing cell can be detected by techniques such as FACS described above. Fixed specimens of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of an antibody.

As a method for screening antibodies using binding activity as an index, a panning method using a phage vector is also suitably used. When screening single domain antibodies, screening can be carried out appropriately according to the following examples.
When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell group, a screening method using a phage vector is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with an appropriate linker sequence to form a single chain Fv (scFv). By inserting a gene encoding an scFv into a phage vector, a phage expressing an scFv on its surface can be obtained. After contacting the phage with a desired antigen, DNA encoding an scFv having a desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this procedure as necessary, scFv having a desired binding activity can be concentrated.

After obtaining a cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. A preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antibody gene. Furthermore, in order to insert one copy of the digested fragment into a vector in the correct direction, it is preferable to insert a restriction enzyme that gives a sticky end. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. At this time, if a gene encoding an antibody constant region (C region) and a gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody whose constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' side of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing the two genes digested with the same combination of restriction enzymes in frame.

To produce an anti-IL-6R monoclonal antibody, an antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for expressing an antibody include, for example, enhancers and promoters. In addition, a suitable signal sequence can be added to the amino terminus so that the expressed antibody is secreted outside the cell. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 1) can be used as a signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide can be secreted outside the cell as a mature polypeptide. Then, a suitable host cell is transformed with this expression vector to obtain a recombinant cell expressing DNA encoding an anti-IL-6R antibody.

Ligand-binding molecule One aspect of the ligand-binding molecule of the present invention is a single domain antibody contained in the ligand-binding molecule that is capable of binding to a ligand, particularly a molecule that is capable of binding to a ligand in an uncleaved state. The term "binding" as used herein generally refers to binding by interactions that are primarily non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Suitable examples of the ligand-binding mode of the ligand-binding molecule of the present invention include, but are not limited to, antigen-antibody reactions in which an antigen-binding region, an antigen-binding molecule, an antibody, and an antibody fragment bind to an antigen.

Note that being able to bind to a ligand means that the ligand-binding molecule is able to bind to the ligand even if the ligand-binding molecule and the ligand are separate molecules, and does not mean that the ligand-binding molecule and the ligand are connected by a covalent bond. For example, being able to bind to a ligand is not said just because a ligand and a ligand-binding molecule are covalently bonded via a linker. Furthermore, being able to weaken the bond with a ligand means that the ability to bind is weakened. For example, when a ligand and a ligand-binding molecule are covalently bonded via a linker, cleavage of the linker cannot be considered to be a weakening of the bond with the ligand. Note that in the present invention, as long as the ligand-binding molecule is able to bind to the ligand, the ligand-binding molecule may be connected to the ligand via a linker or the like.

The ligand-binding molecule of the present invention is limited only in that it binds to a ligand via the single domain antibody contained in the molecule in an uncleaved state, and any structure of molecule can be used as long as it can bind to the desired ligand via the single domain antibody contained in the molecule in an uncleaved state.

One aspect of the ligand-binding molecule of the present invention is a polypeptide that includes a cleavage site. The cleavage site can be cleaved, for example, by an enzyme, reduced by a reducing agent, or photolyzed. The cleavage site can be located anywhere in the polypeptide, as long as it is capable of attenuating the binding of the ligand-binding molecule to the ligand upon cleavage. The polypeptide can include one or more cleavage sites.

In addition, the ligand-binding molecules of the present invention have weaker (i.e., attenuated) ligand binding in the cleaved state compared to the uncleaved state. More specifically, the binding of the single domain antibody in the ligand-binding molecule of the present invention to the ligand is weaker (i.e., attenuated) when the ligand-binding molecule is cleaved than when the ligand-binding molecule is uncleaved. Attenuation of ligand binding can be evaluated by the ligand binding activity of the ligand-binding molecule.

Evaluation of binding activity to ligand The binding activity of a ligand-binding molecule to a ligand can be evaluated by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using the surface plasmon resonance (SPR) phenomenon, and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
The ALPHA screen is carried out by ALPHA technology using two beads, a donor and an acceptor, based on the following principle: A luminescence signal is detected only when a molecule bound to a donor bead interacts with a molecule bound to an acceptor bead and the two beads are in close proximity. A photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, triggers a chemiluminescence reaction in the bead, which ultimately emits light. If there is no interaction between the molecules bound to the donor bead and the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescence reaction occurs.

For example, a biotin-labeled ligand-binding molecule is bound to a donor bead, and a glutathione S-transferase (GST)-tagged ligand is bound to an acceptor bead. In the absence of competing untagged ligand-binding molecules, the ligand-binding molecule and the ligand interact to produce a signal at 520-620 nm. The untagged ligand-binding molecule competes with the interaction between the tagged ligand-binding molecule and the ligand. Relative binding affinity can be determined by quantifying the decrease in fluorescence that results from the competition. It is known to biotinylate ligand-binding molecules such as antibodies using sulfo-NHS-biotin or the like. As a method for tagging a ligand with GST, a method of expressing a GST-fused ligand in a cell or the like that contains a vector capable of expressing a fusion gene in which a polynucleotide encoding the ligand and a polynucleotide encoding GST are fused in frame, and purifying the GST-fused ligand using a glutathione column, or the like, can be appropriately adopted. The resulting signals are suitably analyzed by fitting them to a one-site competition model using nonlinear regression analysis, for example using software such as GRAPHPAD PRISM (GraphPad, San Diego).

One of the substances (ligand) to be observed for interaction is fixed on the gold film of the sensor chip, and light is applied from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass. When the other substance (analyte) to be observed for interaction is poured onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the sensor chip surface changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond dissociates, the position of the signal returns). The Biacore system takes the amount of shift mentioned above, that is, the change in mass on the sensor chip surface, as the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics: binding rate constant (ka) and dissociation rate constant (kd) can be calculated from the curve of the sensorgram, and the dissociation constant (KD) can be calculated from the ratio of these constants. Inhibition measurement methods and equilibrium value analysis methods are also suitable for use in the BIACORE method. An example of an inhibition measurement method is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and an example of an equilibrium value analysis method is described in Methods Enzymol. 2000;323:325-40.

The term "the function of a ligand-binding molecule to bind to a ligand is attenuated" means, for example, that the amount of ligand binding per test ligand-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the control ligand-binding molecule, based on the above-mentioned measurement method. Any suitable index of binding activity may be used, for example, the dissociation constant (KD). When the dissociation constant (KD) is used as an index of binding activity, a larger dissociation constant (KD) of the test ligand-binding molecule to the ligand compared to the dissociation constant (KD) of the control ligand-binding molecule to the ligand indicates that the binding activity of the test ligand-binding molecule to the ligand is weaker than that of the control ligand-binding molecule. The function of binding to a ligand is weakened, for example, when the dissociation constant (KD) of the test ligand-binding molecule for the ligand is at least 2-fold, preferably at least 5-fold, at least 10-fold, and particularly preferably at least 100-fold, compared to the dissociation constant (KD) of the control ligand-binding molecule for the ligand.
The control ligand-binding molecule can be, for example, an uncleaved form of the ligand-binding molecule.

In one embodiment of the present invention, the cleavage site of the ligand-binding molecule of the present invention is cleaved, thereby releasing the ligand from the ligand-binding molecule. Here, if the ligand is fused to a portion of the ligand-binding molecule via a linker and the linker does not have a cleavage site, the ligand will be released while still connected to the portion of the ligand-binding molecule via the linker (see, for example, Figures 2 and 4). In this way, even if the ligand is released together with a portion of the ligand-binding molecule, it can be said that the ligand has been released from the ligand-binding molecule as long as there is a portion of the ligand-binding molecule that can no longer stably interact with the ligand.

A method for detecting the release of a ligand from a ligand-binding molecule due to cleavage at the cleavage site includes a method of detecting the ligand using a ligand-detecting antibody or the like that recognizes the ligand. When the ligand-binding molecule is an antibody fragment, the ligand-detecting antibody preferably binds to the same epitope as the ligand-binding molecule. Detection of the ligand using a ligand-detecting antibody can be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using the surface plasmon resonance (SPR) phenomenon, and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
For example, when detecting the release of a ligand using Octet, a ligand-detecting antibody that recognizes the ligand is biotinylated, contacted with a biosensor, and then the binding to the ligand in the sample is measured to detect the release of the ligand. Specifically, the amount of the ligand is measured using a ligand detection antibody for a sample containing a ligand-binding molecule and a ligand before or after protease treatment, and the amount of the ligand detected in the sample before and after protease treatment is compared to detect the release of the ligand. In addition, the amount of the ligand is measured using a ligand detection antibody for a sample containing a protease, a ligand-binding molecule, and a ligand, and a sample containing a ligand-binding molecule and a ligand without protease, and the amount of the ligand detected in the sample with and without protease is compared to detect the release of the ligand. More specifically, the release of the ligand can be detected by the method in the Examples of the present application. When a ligand-binding molecule is fused with a ligand to form a fusion protein, the amount of the ligand is measured using a ligand detection antibody for a sample containing a fusion protein before or after protease treatment, and the amount of the ligand detected in the sample before and after protease treatment is compared to detect the release of the ligand. In addition, the amount of ligand is measured using a ligand detection antibody for a sample containing a protease and a fusion protein and a sample containing a fusion protein without a protease, and the amount of ligand detected in the samples with and without the protease is compared to detect the release of the ligand. More specifically, the release of the ligand can be detected by the method described in the Examples of the present application.

In an embodiment in which the biological activity of the ligand is inhibited when it binds to the ligand-binding molecule, the release of the ligand can be detected by measuring the biological activity of the ligand in a sample as a method for detecting release from the ligand-binding molecule. Specifically, the release of the ligand can be detected by measuring and comparing the biological activity of the ligand for a sample containing the ligand-binding molecule and the ligand before or after protease treatment. The release of the ligand can also be detected by measuring and comparing the biological activity of the ligand for a sample containing a protease, the ligand-binding molecule, and the ligand, and for a sample containing the ligand-binding molecule and the ligand without the protease. When the ligand-binding molecule is fused with the ligand to form a fusion protein, the release of the ligand can be detected by measuring and comparing the biological activity of the ligand for a sample containing the fusion protein before or after protease treatment. The release of the ligand can also be detected by measuring and comparing the biological activity of the ligand for a sample containing the protease and the fusion protein, and for a sample containing the fusion protein without the protease.

In one embodiment of the invention, the cleavage site contains a protease cleavage sequence and is cleaved by a protease.

Protease Cleavage Sequence A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by the target tissue-specific protease in an aqueous solution.
From the viewpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed or more specifically activated in the target tissue/cells to be treated.
Specific examples of protease cleavage sequences include target sequences that are specifically hydrolyzed by the above-mentioned cancer tissue-specific proteases and inflammatory tissue-specific proteases disclosed in International Publication WO2013/128194, International Publication WO2010/081173, International Publication WO2009/025846, etc. Artificially modified sequences, such as those obtained by introducing appropriate amino acid mutations into target sequences that are specifically hydrolyzed by known proteases, can also be used. Protease cleavage sequences identified by methods known to those skilled in the art, such as those described in Nature Biotechnology 19, 661-667 (2001), may also be used.
Furthermore, a naturally occurring protease cleavage sequence may be used. For example, a sequence in a protein that undergoes protease cleavage and changes its molecular shape upon cleavage by a protease, such as TGF-β being converted to its latent form upon cleavage by a protease, may be used.

Examples of protease cleavage sequences include, but are not limited to, those described in International Publication No. WO2015/116933, International Publication No. WO2015/048329, International Publication No. WO2016/118629, International Publication No. WO2016/179257, International Publication No. WO2016/179285, International Publication No. WO2016/179335, International Publication No. WO2016/179003, International Publication No. WO2016/046778, International Publication No. WO2016/014974, U.S. Patent Publication No. US2016/0289324, U.S. Patent Publication No. US2016/0311903, PNAS (2000) 97: 7754-7759., Biochemical Journal (2010) 426: 219-228., Beilstein J Nanotechnol. (2016) 7: 364-373. can be used.
More preferably, the protease cleavage sequence is an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease, as described above. Among the amino acid sequences that are specifically hydrolyzed by a target tissue-specific protease, the following amino acid sequences are preferred:
LSGRSDNH (SEQ ID NO:2, MT-SP1, cleavable by uPA)
PLGLAG (SEQ ID NO: 3, cleavable by MMP-2 and MMP-9)
VPLSLTMG (SEQ ID NO: 4, cleavable by MMP-7)
The following sequences can also be used as protease cleavage sequences:
TSTSGRSANPRG (SEQ ID NO:5, MT-SP1, cleavable by uPA)
ISSGLLSGRSDNH (SEQ ID NO:6, MT-SP1, cleavable by uPA)
AVGLLAPPGGLSGRSDNH (SEQ ID NO: 7, MT-SP1, cleavable by uPA)
GAGVPMSMRGGAG (SEQ ID NO:8, cleavable by MMP-1)
GAGIPVSLRSGAG (SEQ ID NO:9, cleavable by MMP-2)
GPLGIAGQ (SEQ ID NO: 10, cleavable by MMP-2)
GGPLGMLSQS (SEQ ID NO: 11, cleavable by MMP-2)
PLGLWA (SEQ ID NO:12, cleavable by MMP-2)
GAGRPFSMIMGAG (SEQ ID NO: 13, cleavable by MMP-3)
GAGVPLSLTMGAG (SEQ ID NO: 14, cleavable by MMP-7)
GAGVPLSLYSGAG (SEQ ID NO:15, cleavable by MMP-9)
AANLRN (SEQ ID NO: 16, cleavable by MMP-11)
AQAYVK (SEQ ID NO: 17, cleavable by MMP-11)
AANYMR (SEQ ID NO:18, cleavable by MMP-11)
AAALTR (SEQ ID NO: 19, cleavable by MMP-11)
AQNLMR (SEQ ID NO:20, cleavable by MMP-11)
AANYTK (SEQ ID NO:21, cleavable by MMP-11)
GAGPQGLAGQRGIVAG (SEQ ID NO:22, cleavable by MMP-13)
PRFKIIGG (SEQ ID NO:23, pro-cleavable by urokinase)
PRFRIIGG (SEQ ID NO:24, pro-cleavable by urokinase)
GAGSGRSAG (SEQ ID NO:25, cleavable by uPA)
SGRSA (SEQ ID NO:26, cleavable by uPA)
GSGRSA (SEQ ID NO:27, cleavable by uPA)
SGKSA (SEQ ID NO:28, cleavable by uPA)
SGRSS (SEQ ID NO:29, cleavable by uPA)
SGRRA (SEQ ID NO:30, cleavable by uPA)
SGRNA (SEQ ID NO:31, cleavable by uPA)
SGRKA (SEQ ID NO:32, cleavable by uPA)
QRGRSA (SEQ ID NO:33, cleavable by tPA)
GAGSLLKSRMVPNFNAG (SEQ ID NO:34, cleavable by cathepsin B)
TQGAAA (SEQ ID NO:35, cleavable by cathepsin B)
GAAAAAA (SEQ ID NO: 36, cleavable by cathepsin B)
GAGAAG (SEQ ID NO:37, cleavable by cathepsin B)
AAAAAG (SEQ ID NO:38, cleavable by cathepsin B)
LCGAAI (SEQ ID NO:39, cleavable by cathepsin B)
FAQALG (SEQ ID NO:40, cleavable by cathepsin B)
LLQANP (SEQ ID NO:41, cleavable by cathepsin B)
LAAANP (SEQ ID NO:42, cleavable by cathepsin B)
LYGAQF (SEQ ID NO: 43, cleavable by cathepsin B)
LSQAQG (SEQ ID NO:44, cleavable by cathepsin B)
ASAASG (SEQ ID NO:45, cleavable by cathepsin B)
FLGASL (SEQ ID NO:46, cleavable by cathepsin B)
AYGATG (SEQ ID NO:47, cleavable by cathepsin B)
LAQATG (SEQ ID NO:48, cleavable by cathepsin B)
GAGSGVVIATVIVITAG (SEQ ID NO:49, cleavable by cathepsin L)
APMAEGGG (SEQ ID NO: 50, cleavable by meprin α and meprin β)
EAQGDKII (SEQ ID NO: 51, cleavable by meprin α and meprin β)
LAFSDAGP (SEQ ID NO: 52, cleavable by meprin α and meprin β)
YVADAPK (SEQ ID NO: 53, cleavable by meprin α and meprin β)
RRRRR (SEQ ID NO:54, cleavable by furin)
RRRRRR (SEQ ID NO:55, cleavable by furin)
GQSSRHRRAL (SEQ ID NO:56, cleavable by furin)
SSRHRRALD (SEQ ID NO:57)
RKSSIIIRMRDVVL (SEQ ID NO:58, cleavable by plasminogen)
SSSFDKGKYKKGDDA (SEQ ID NO: 59, cleavable by Staphylokinase)
SSSFDKGKYKRGDDA (SEQ ID NO: 60, cleavable by Staphylokinase)
IEGR (SEQ ID NO:61, cleavable by Factor IXa)
IDGR (SEQ ID NO:62, cleavable by Factor IXa)
GGSIDGR (SEQ ID NO: 63, cleavable by Factor IXa)
GPQGIAGQ (SEQ ID NO: 64, cleavable by collagenase)
GPQGLLGA (SEQ ID NO: 65, cleavable by collagenase)
GIAGQ (SEQ ID NO: 66, cleavable by collagenase)
GPLGIAG (SEQ ID NO: 67, cleavable by collagenase)
GPEGLRVG (SEQ ID NO: 68, cleavable by collagenase)
YGAGLGVV (SEQ ID NO: 69, cleavable by collagenase)
AGLGVVER (SEQ ID NO: 70, cleavable by collagenase)
AGLGISST (SEQ ID NO: 71, cleavable by collagenase)
EPQALAMS (SEQ ID NO: 72, cleavable by collagenase)
QALAMSAI (SEQ ID NO: 73, cleavable by collagenase)
AAYHLVSQ (SEQ ID NO: 74, cleavable by collagenase)
MDAFLESS (SEQ ID NO: 75, cleavable by collagenase)
ESLPVVAV (SEQ ID NO: 76, cleavable by collagenase)
SAPAVESE (SEQ ID NO: 77, cleavable by collagenase)
DVAQFVLT (SEQ ID NO: 78, cleavable by collagenase)
VAQFVLTE (SEQ ID NO: 79, cleavable by collagenase)
AQFVLTEG (SEQ ID NO: 80, cleavable by collagenase)
PVQPIGPQ (SEQ ID NO: 81, cleavable by collagenase)
LVPRGS (SEQ ID NO:82, cleavable by Thrombin)
TSGSGRSANARG (SEQ ID NO:170, cleavable by uPA and MT-SP1)
TSQSGRSANQRG (SEQ ID NO:171, cleavable by uPA and MT-SP1)
TSPSGRSAYPRG (SEQ ID NO: 172, cleavable by uPA and MT-SP1)
TSGSGRSATPRG (SEQ ID NO: 173, cleavable by uPA and MT-SP1)
TSQSGRSATPRG (SEQ ID NO:174, cleavable by uPA and MT-SP1)
TSASGRSATPRG (SEQ ID NO:175, cleavable by uPA and MT-SP1)
TSYSGRSAVPRG (SEQ ID NO: 176, cleavable by uPA and MT-SP1)
TSYSGRSANFRG (SEQ ID NO:177, cleavable by uPA and MT-SP1)
TSSSGRSATPRG (SEQ ID NO: 178, cleavable by uPA and MT-SP1)
TSTTGRSASPRG (SEQ ID NO: 179, cleavable by uPA and MT-SP1)

The sequences shown in Table 1 can also be used as protease cleavage sequences.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:788)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:789)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 790)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, X8 is an amino acid selected from A, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:791)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:792)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:793)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and W; X7 is an amino acid selected from A, D, E, F, G, H, I, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:794)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is X is an amino acid selected from A, D, F, G, L, M, P, Q, V and W; X is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 795)
X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:796)
X1 to X8 each represent an amino acid, where X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; and X8 is an amino acid selected from H, V and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO:797)
X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; and X8 is an amino acid selected from H, P, V, and Y.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 798)
X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 799)
X1 to X9 each represent an amino acid, X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 being an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 being an amino acid selected from A, D, E, F, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 800)
X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, X8 is an amino acid selected from A, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 801)
X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 802)
X1 to X9 each represent an amino acid, X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 being R; X5 being an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W and Y; X6 being an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 being an amino acid selected from A, D, E, F, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 803)
X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and W; X7 is an amino acid selected from A, D, E, F, G, H, I, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 804)
X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 805)
X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 806)
X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

The following protease cleavage sequences may also be used:
X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 807)
X is an amino acid selected from A and E; X is an amino acid selected from H, P, V, and Y; and X is an amino acid selected from A, G, H, I, L, and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 808)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 809)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 810)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 811)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 812)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 813)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and W. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 814)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 815)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 816)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; and X8 is an amino acid selected from H, V and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 817)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V and Y.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 818)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 819)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 820)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 821)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 822)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 823)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and W. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 824)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 825)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W; X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 826)
X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; X8 is an amino acid selected from H, V and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

The following protease cleavage sequences may also be used:
X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 827)
X7 is an amino acid selected from N and V; X8 is an amino acid selected from H, P, V and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

In addition to using the above protease cleavage sequences, new protease cleavage sequences may be obtained by screening. For example, new protease cleavage sequences can be searched for by changing the interaction between the cleavage sequence and the active residues/recognition residues of the enzyme based on the results of crystal structure analysis of known protease cleavage sequences. Also, new protease cleavage sequences can be searched for by modifying amino acids in known protease cleavage sequences and confirming the interaction with the protease. As another example, sequences cleaved by proteases can be searched for by displaying a peptide library using an in vitro display method such as phage display or ribosome display, or by confirming the interaction with the protease using a peptide array fixed to a chip or beads.
The interaction between a protease cleavage sequence and a protease can be confirmed by confirming protease cleavage in vitro or in vivo.

For example, any of the polypeptides containing the protease cleavage sequences exemplified in Table 1 are useful as protease substrates that are hydrolyzed by the action of a protease. The protease substrates shown in SEQ ID NOs: 788-827 and the protease substrates in Table 1 can be used as a library for selecting those with properties according to the purpose when incorporated into a ligand-binding molecule. Specifically, the protease sensitivity of a ligand-binding molecule can be evaluated in order to selectively cleave the molecule with a protease localized at the lesion. After being administered to a living body, a ligand-binding molecule bound to a ligand may reach the lesion through contact with various proteases. Therefore, it is desirable for the molecule to be sensitive to proteases localized at the lesion while being resistant to other proteases as much as possible. In order to select a desired protease cleavage sequence according to the purpose, the protease resistance can be known by comprehensively analyzing the sensitivity of each protease substrate to various proteases in advance. Based on the obtained protease resistance spectrum, a protease cleavage sequence with the required sensitivity and resistance can be found.
Alternatively, a ligand-binding molecule incorporating a protease cleavage sequence reaches the lesion not only through the enzymatic action of a protease but also through various environmental stresses such as changes in pH, temperature, redox stress, etc. Based on information comparing the resistance of each protease substrate to such external factors, a protease cleavage sequence having desirable properties according to the purpose can be selected.

In one embodiment of the invention, the protease cleavage sequence further comprises a flexible linker at either or both ends. The flexible linker at one end of the protease cleavage sequence can be referred to as a first flexible linker and the flexible linker at the other end can be referred to as a second flexible linker. In certain embodiments, the protease cleavage sequence and the flexible linker comprise one of the following formulas:
(Protease cleavage sequence)
(first flexible linker)-(protease cleavage sequence)
(protease cleavage sequence)-(second flexible linker)
(first flexible linker)-(protease cleavage sequence)-(second flexible linker)
The flexible linker in this embodiment is preferably a peptide linker. The first and second flexible linkers are independently and optionally present, and are the same or different flexible linkers that contain at least one flexible amino acid (such as Gly). For example, a sufficient number of residues (amino acids selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, Ala, especially Gly and Ser, especially Gly, etc.) are included so that the protease cleavage sequence has the desired protease accessibility.

Flexible linkers suitable for use at both ends of the protease cleavage sequence typically improve the accessibility of the protease to the protease cleavage sequence and increase the cleavage efficiency of the protease. Suitable flexible linkers can be easily selected and are of different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, or 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, including 3 to 12 amino acids. In some embodiments of the invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

Examples of flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS: SEQ ID NO:92)n, and (GGGS: SEQ ID NO:83)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
Of these, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and thus tend to function as neutral tethers between components.
Examples of flexible linkers made of glycine-serine polymers include, but are not limited to,
Ser
Gly-Ser (GS)
Ser-Gly (SG)
Gly-Gly-Ser (GGS)
Gly-Ser-Gly (GSG)
Ser-Gly-Gly (SGG)
Gly-Ser-Ser (GSS)
Ser-Ser-Gly (SSG)
Ser-Gly-Ser (SGS)
Gly.Gly.Gly.Ser (GGGS, SEQ ID NO:83)
Gly.Gly.Ser.Gly (GGSG, SEQ ID NO: 84)
Gly.Ser.Gly.Gly (GSGG, SEQ ID NO: 85)
Ser.Gly.Gly.Gly (SGGG, SEQ ID NO: 86)
Gly.Ser.Ser.Gly (GSSG, SEQ ID NO:87)
Gly.Gly.Gly.Gly.Gly.Ser (GGGGS, SEQ ID NO:88)
Gly.Gly.Gly.Ser.Gly (GGGSG, SEQ ID NO: 89)
Gly.Gly.Ser.Gly.Gly (GGSGG, SEQ ID NO:90)
Gly.Ser.Gly.Gly.Gly.Gly (GSGGG, SEQ ID NO:91)
Gly.Ser.Gly.Gly.Ser (GSGGS, SEQ ID NO:92)
Ser.Gly.Gly.Gly.Gly.Gly (SGGGG, SEQ ID NO:93)
Gly.Ser.Ser.Gly.Gly (GSSGG, SEQ ID NO:94)
Gly.Ser.Gly.Ser.Gly.Gly (GSGSG, SEQ ID NO:95)
Ser.Gly.Gly.Ser.Gly (SGGSG, SEQ ID NO:96)
Gly.Ser.Ser.Ser.Gly (GSSSG, SEQ ID NO:97)
Gly.Gly.Gly.Gly.Gly.Gly.Gly.Ser (GGGGGS, SEQ ID NO:98)
Ser Gly Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 99)
Gly.Gly.Gly.Gly.Gly.Gly.Gly.Gly.Ser (GGGGGGS, SEQ ID NO:100)
Ser Gly Gly Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 101)
(Gly.Gly.Gly.Gly.Gly.Ser (GGGGS, SEQ ID NO:88)
(Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 93)
[n is an integer of 1 or more], etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

In some embodiments of the present invention, cleavage of the cleavage site/protease cleavage sequence in the ligand-binding molecule disrupts the single domain antibody in the ligand-binding molecule, thereby reducing binding to the ligand.

In one embodiment of the present invention, the cleavage site/protease cleavage sequence is introduced into a single domain antibody in a ligand binding molecule. In some more specific embodiments, the cleavage site/protease cleavage sequence is introduced at the position of residues forming a loop structure in the single domain antibody and residues close to the loop structure. The loop structure in the single domain antibody refers to the portion of the single domain antibody that does not form a secondary structure such as an α-helix or β-sheet.

In an embodiment in which the single-domain antibody is a VHH or single-domain VH antibody, the positions of the residues forming the loop structure and the residues close to the loop structure are specifically: single-domain antibody, amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), It can refer to the range from amino acid number 55 (Kabat numbering) to amino acid number 69 (Kabat numbering), amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), amino acid number 95 (Kabat numbering) to amino acid number 102 (Kabat numbering), and amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering).

In embodiments where the single domain antibody is a VHH or a single domain VH antibody, the cleavage site/protease cleavage sequence is introduced into one or more positions within one or more sequences selected from the following sequences of the single domain antibody:
Single domain antibody: sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering); single domain antibody: sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering); single domain antibody: sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering); single domain antibody: sequence from amino acid 55 (Kabat numbering) to the sequence up to amino acid number 69 (Kabat numbering), single domain antibody, sequence from amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), single domain antibody, sequence from amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 102 (Kabat numbering), single domain antibody, sequence from amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering).

In an embodiment in which the single domain antibody is a single domain VL antibody, the positions of the residues forming the loop structure and the residues close to the loop structure can specifically refer to the ranges from amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering), from amino acid 24 (Kabat numbering) to amino acid 34 (Kabat numbering), from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), from amino acid 50 (Kabat numbering) to amino acid 56 (Kabat numbering), from amino acid 89 (Kabat numbering) to amino acid 97 (Kabat numbering), and from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering).

In embodiments where the single domain antibody is a single domain VL antibody, the cleavage site/protease cleavage sequence is introduced into one or more positions within one or more sequences selected from the following sequences of the single domain antibody:
A single domain antibody sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering), a single domain antibody sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering), a single domain antibody sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering), a single domain antibody sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering), a single domain antibody sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering), a single domain antibody sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering), a single domain antibody sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering).

The cleavage sites/protease cleavage sequences can be provided in multiple locations within a ligand-binding molecule, for example, in a single domain antibody within the ligand-binding molecule.

In some embodiments of the present invention, the biological activity of a ligand is inhibited by binding to an uncleaved ligand-binding molecule. Non-limiting examples of embodiments in which the biological activity of a ligand is inhibited include, for example, embodiments in which the binding of an uncleaved ligand-binding molecule to a ligand substantially or significantly interferes with or competes with the binding of the ligand to its binding partner. When an antibody or a fragment thereof having ligand-neutralizing activity is used as the ligand-binding molecule, the biological activity of the ligand can be inhibited by the ligand-binding molecule binding to the ligand exerting its neutralizing activity.

In one embodiment of the present invention, it is preferable that the uncleaved ligand-binding molecule can sufficiently neutralize the biological activity of the ligand by binding to the ligand. In other words, it is preferable that the biological activity of the ligand bound to the uncleaved ligand-binding molecule is lower than the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. Although not limited thereto, for example, the biological activity of the ligand bound to the uncleaved ligand-binding molecule may be 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. By sufficiently neutralizing the biological activity of the ligand, it is expected that when the ligand-binding molecule is administered, the ligand will not exert its biological activity before reaching the target tissue.

In one embodiment of the present invention, the binding activity of the cleaved ligand-binding molecule to the ligand is preferably lower than the binding activity of the in vivo natural binding partner (e.g., a natural receptor for the ligand) to the ligand. For example, the binding activity of the cleaved ligand-binding molecule to the ligand is 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, and particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the binding amount of the natural binding partner to the ligand in the body (per unit binding partner), but is not limited thereto. As an index of binding activity, any desired index may be used, for example, the dissociation constant (KD). When the dissociation constant (KD) is used as an evaluation index of binding activity, a larger dissociation constant (KD) of the cleaved ligand-binding molecule for the ligand than the dissociation constant (KD) of the natural binding partner in vivo indicates that the binding activity of the cleaved ligand-binding molecule for the ligand is weaker than that of the natural binding partner in vivo. The dissociation constant (KD) of the cleaved ligand-binding molecule for the ligand is, for example, 1.1 times or more, preferably 1.5 times or more, 2 times or more, 5 times or more, 10 times or more, and particularly preferably 100 times or more than the dissociation constant (KD) of the natural binding partner in vivo. Since the cleaved ligand-binding molecule has only low binding activity to the ligand or has almost no binding activity to the ligand, it is expected that the ligand-binding molecule will release the ligand after cleavage and will be prevented from rebinding to another ligand molecule.

After the ligand-binding molecule is cleaved, it is desirable that the inhibited biological activity of the ligand is restored. It is desirable that the binding of the cleaved ligand-binding molecule to the ligand is attenuated, so that the function of the ligand-binding molecule in inhibiting the biological activity of the ligand is also attenuated. The biological activity of the ligand can be confirmed by one of skill in the art using known methods, such as methods for detecting binding between the ligand and its binding partner.

In some embodiments of the invention, the uncleaved ligand-binding molecule has a longer half-life than the single domain antibody alone. Examples of embodiments in which the serum half-life of the ligand-binding molecule is longer than the serum half-life of the single domain antibody alone include, but are not limited to, increasing the molecular weight of the ligand-binding molecule, fusing the single domain antibody to a moiety that has FcRn binding ability, fusing the single domain antibody to a moiety that has albumin binding ability, or fusing the single domain antibody to a moiety that is PEGylated.

In the present invention, the half-life of a single domain antibody alone and a ligand-binding molecule is preferably compared based on the blood half-life in humans. If it is difficult to measure the blood half-life in humans, the blood half-life in humans can be predicted based on the blood half-life in mice (e.g., normal mice, transgenic mice expressing human antigens, transgenic mice expressing human FcRn, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

One embodiment for making the blood half-life of a ligand-binding molecule longer than that of a single domain antibody alone is to increase the molecular weight of the ligand-binding molecule. In a preferred embodiment, the molecular weight of the ligand-binding molecule is 60 kDa or more. Molecules with a molecular weight of 60 kDa or more are generally less likely to be cleared by the kidney when present in the blood, and are more likely to have a long blood half-life (see J Biol Chem. 1988 Oct 15;263(29):15064-70).

One embodiment for making the blood half-life of a ligand-binding molecule longer than that of a single domain antibody alone is to fuse a single domain antibody with a moiety having FcRn-binding ability. The moiety having FcRn-binding ability usually has an FcRn-binding region. The FcRn-binding region refers to a region having binding ability to FcRn, and any structure can be used as long as it has binding ability to FcRn.
The transporter containing the FcRn-binding region can return to plasma after being taken up into cells via the FcRn salvage pathway. For example, the relatively long plasma retention of IgG molecules (slow disappearance) is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions in the endosome. IgG molecules that cannot bind to FcRn proceed to lysosomes where they are degraded, while IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma, returning to plasma.
The FcRn-binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn-binding region is the Fc region of an antibody. However, since a region capable of binding to a polypeptide having FcRn-binding ability, such as albumin or IgG, can indirectly bind to FcRn via albumin, IgG, or the like, the FcRn-binding region of the present invention may be a region that binds to such a polypeptide having FcRn-binding ability.

The binding activity of the FcRn-binding region of the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described above in the section on binding activity, and the conditions can be appropriately determined by those skilled in the art. The binding activity to human FcRn can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate), apparent kd (Apparent dissociation), or the like. These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc. can be used.

The conditions for measuring the binding activity of the FcRn-binding region to FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009/125825, the measurement can be performed under conditions of MES buffer and 37°C. Furthermore, the binding activity of the FcRn-binding region of the present invention to FcRn can be measured by a method known to those skilled in the art, for example, using Biacore (GE Healthcare). The binding activity of the FcRn-binding region to FcRn can be evaluated by flowing FcRn, the FcRn-binding region, or the transport moiety containing the FcRn-binding region as an analyte through a chip on which the FcRn-binding region or the transport moiety containing the FcRn-binding region is immobilized, or FcRn is immobilized.

As the pH used as the measurement condition, the binding affinity between the FcRn-binding region and FcRn may be evaluated at any pH between pH 4.0 and pH 6.5. Preferably, a pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn-binding region and human FcRn. As the temperature used as the measurement condition, the binding affinity between the FcRn-binding region and FcRn may be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the FcRn-binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn-binding region and FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

An example of an FcRn binding region is, but is not limited to, an IgG antibody Fc region. When using an IgG antibody Fc region, the type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region containing one of the amino acid sequences shown in SEQ ID NOs: 103, 104, 105, and 106 can be used.

Furthermore, not only the Fc region of a natural IgG antibody, but also a modified Fc region in which one or more amino acids have been substituted can be used, so long as it has FcRn-binding ability.
For example, the following amino acids in the IgG antibody Fc region are listed below (EU numbering): 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, and 314 It is possible to use a modified Fc region comprising an amino acid sequence in which at least one amino acid selected from among positions 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 has been replaced with another amino acid.

More specifically, EU numbering in the IgG antibody Fc region
Amino acid substitution at position 237, replacing Gly with Met;
Amino acid substitution of Pro at position 238 to Ala;
Amino acid substitution at position 239 replacing Ser with Lys;
Amino acid substitution at position 248, replacing Lys with Ile;
an amino acid substitution of Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr;
an amino acid substitution at position 252 replacing Met with Phe, Trp, or Tyr;
Amino acid substitution at position 254 replacing Ser with Thr;
Amino acid substitution of Arg at position 255 to Glu;
an amino acid substitution of Thr at position 256 with Asp, Glu, or Gln;
an amino acid substitution of Pro at position 257 with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val;
Amino acid substitution at position 258, replacing Glu with His;
Amino acid substitution of Asp at position 265 to Ala;
an amino acid substitution of Asp at position 270 with Phe;
Amino acid substitution of Asn at position 286 with Ala or Glu;
Amino acid substitution of Thr at position 289 to His;
an amino acid substitution of Asn at position 297 to Ala;
an amino acid substitution at position 298, replacing Ser with Gly;
Amino acid substitution of Val at position 303 to Ala;
Amino acid substitution of Val to Ala at position 305;
an amino acid substitution replacing Thr at position 307 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;
an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr;
an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro, or Arg;
an amino acid substitution of Gln at position 311 with Ala, His, or Ile;
an amino acid substitution of Asp at position 312 with Ala or His;
Amino acid substitution of Leu at position 314 with Lys or Arg;
an amino acid substitution of Asn at position 315 with Ala or His;
Amino acid substitution of Lys at position 317 to Ala;
an amino acid substitution of Asn at position 325 with Gly;
an amino acid substitution at position 332 replacing Ile with Val;
Amino acid substitution at position 334, replacing Lys with Leu;
Amino acid substitution at position 360, replacing Lys with His;
an amino acid substitution of Asp at position 376 with Ala;
Amino acid substitution of Glu at position 380 to Ala;
Amino acid substitution of Glu at position 382 to Ala;
an amino acid substitution at position 384 replacing Asn or Ser with Ala;
an amino acid substitution of Gly at position 385 with Asp or His;
an amino acid substitution of Gln at position 386 with Pro;
Amino acid substitution of Pro at position 387 with Glu;
an amino acid substitution of Asn at position 389 with Ala or Ser;
Amino acid substitution of Ser at position 424 to Ala;
an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;
Amino acid substitution at position 433, replacing His with Lys;
an amino acid substitution replacing Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr; and
It is possible to use a modified Fc region comprising at least one amino acid substitution selected from the amino acid substitution at position 436 replacing Tyr or Phe with His.

From another perspective, the EU numbering in the IgG antibody Fc region
Met at amino acid 237;
Ala at amino acid 238;
Lys at amino acid 239;
Ile at amino acid 248;
Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at amino acid position 250;
Phe, Trp, or Tyr at amino acid 252;
Thr at amino acid 254;
Glu at amino acid 255,
Asp, Glu, or Gln at amino acid position 256;
Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257;
His at amino acid 258;
Ala at amino acid 265;
Phe at amino acid 270,
Ala or Glu at amino acid 286;
His at amino acid 289,
Ala at amino acid 297;
Gly at amino acid 298;
Ala at amino acid 303;
Ala at amino acid 305;
Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid 307;
Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid 308;
Ala, Asp, Glu, Pro, or Arg at amino acid position 309;
Ala, His, or Ile at amino acid 311;
Ala or His at amino acid 312;
Lys or Arg at amino acid 314;
Ala or His at amino acid 315;
Ala at amino acid 317;
Gly at amino acid 325;
Val at amino acid 332;
Leu at amino acid 334,
His at amino acid 360,
Ala at amino acid 376;
Ala at amino acid 380;
Ala at amino acid 382;
Ala at amino acid 384;
Asp or His at amino acid 385;
Pro at amino acid 386,
Glu at amino acid 387,
Ala or Ser at amino acid 389;
Ala at amino acid 424;
Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid 428;
Lys at amino acid 433;
Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and
His at amino acid 436
It is possible to use an Fc region comprising at least one amino acid selected from the following:

In one embodiment of the present invention, the half-life of a ligand-binding molecule in the blood is longer than that of a single domain antibody alone, and this can be achieved by fusing the single domain antibody with a moiety that binds to albumin. Albumin is not excreted by the kidney and has FcRn binding ability, so it has a long half-life in the blood of 17 to 19 days (J Clin Invest. 1953 Aug; 32(8): 746-768.). It has been reported that proteins bound to albumin therefore become bulky and are able to indirectly bind to FcRn, thereby increasing their half-life in the blood (Antibodies 2015, 4(3), 141-156).

Furthermore, as an embodiment for extending the blood half-life of a ligand-binding molecule beyond that of a single domain antibody, there is a method of fusing a single domain antibody with a PEGylated portion. It is believed that PEGylation of a protein increases the protein's bulkiness and at the same time inhibits degradation by proteases in the blood, thereby extending the protein's blood half-life (J Pharm Sci. 2008 Oct;97(10):4167-83.).

In some embodiments of the invention, the ligand binding molecule comprises an antibody Fc region. In one specific embodiment, the ligand binding molecule comprises the CH2 and CH3 domains of a human IgG antibody. In one specific embodiment, the ligand binding molecule comprises a portion of a human IgG1 antibody heavy chain extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, with the proviso that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.

Ligand : As used herein, the term "ligand" refers to a biologically active molecule that typically functions by interacting with a cell surface receptor and thereby stimulating, inhibiting, or otherwise modulating a biological signaling pathway that typically appears to be involved in a signaling pathway within the cell that bears the receptor.

In this specification, the term "ligand" includes a desired molecule that exerts biological activity by interacting with a biomolecule. For example, the term "ligand" does not only mean a molecule that interacts with a receptor, but also includes a molecule that exerts biological activity by interacting with the molecule, and for example, a receptor that interacts with the molecule and binding fragments thereof are also included in the ligand. For example, a protein that contains a ligand-binding site of a protein known as a receptor, or a site where the receptor interacts with another molecule, is included in the ligand in the present invention. Specifically, soluble receptors, soluble fragments of receptors, the extracellular domain of a transmembrane receptor, and polypeptides containing them are included in the ligand in the present invention.

The ligands of the invention typically exert their desired biological activity by binding to one or more binding partners. The binding partner of a ligand can be an extracellular, intracellular, or transmembrane protein. In one embodiment, the binding partner of a ligand is an extracellular protein, such as a soluble receptor. In another embodiment, the binding partner of a ligand is a membrane-bound receptor.
A ligand of the invention can specifically bind to its binding partner with a dissociation constant (KD) of less than or equal to 10 μM, 1 μM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 0.5 pM, or 0.1 pM.

Examples of biologically active molecules include, but are not limited to, cytokines, chemokines, polypeptide hormones, growth factors, apoptosis inducers, PAMPs, DAMPs, nucleic acids, or fragments thereof. In particular embodiments, the ligand may be an interleukin, an interferon, a hematopoietic factor, a TNF superfamily, a chemokine, a cell growth factor, a TGF-β family, a myokine, an adipokines, or a neurotrophic factor. In more specific embodiments, the ligand may be CXCL10, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-g, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1 (Interleukin-1 receptor, type I), IL-1R2 (Interleukin-1 receptor, type II), IL-1RAcP (Interleukin-1 receptor accessory protein), or IL-1Ra (protein accession no. NP_776214, mRNA accession no. NM_173842.2).

Chemokines are a family of homogeneous serum proteins between 7 and 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines have four characteristic cysteines (Cys) and are classified into the CXC, or alpha, CC, or beta, C, or gamma, and CX3C, or delta, chemokine classes, depending on the motif represented by the first two cysteines. Two disulfide bonds are formed between the first and third cysteines and between the second and fourth cysteines. In general, disulfide bridges are considered necessary, and Clark-Lewis and coworkers reported that, at least for CXCL10, disulfide bonds are crucial for chemokine activity (Clark-Lewis et al., J. Biol. Chem. 269:16075-16081, 1994). The only exception to having four cysteines is lymphotactin, which has only two cysteine residues and thus manages to maintain a functional structure with only one disulfide bond.
The CXC or alpha subfamily has been further divided into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines, depending on the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine (see, e.g., Clark-Lewis, supra, and Belperio et al., "CXC Chemokines in Angiogenesis," J. Leukoc. Biol. 68:1-8, 2000).

Interferon-inducible protein-10 (IP-10 or CXCL10) is induced by interferon-γ and TNF-α and is produced by keratinocytes, endothelial cells, fibroblasts, and monocytes. IP-10 is thought to play a role in the recruitment of activated T cells to sites of tissue inflammation (Dufour, et al., "IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking," J Immunol., 168:3195-204, 2002). In addition, IP-10 may play a role in hypersensitivity reactions. It may also play a role in the development of inflammatory demyelinating neuropathies (Kieseier, et al., "Chemokines and chemokine receptors in inflammatory demyelinating neuropathies: a central role for IP-10," Brain 125:823-34, 2002).

Studies have shown that IP-10 may be useful in stem cell engraftment following transplantation (Nagasawa, T., Int. J. Hematol. 72:408-11, 2000), stem cell mobilization (Gazitt, Y., J. Hematother Stem Cell Res 10:229-36, 2001; Hattori et al., Blood 97:3354-59, 2001), and enhancing antitumor immunity (Nomura et al., Int. J. Cancer 91:597-606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, the biological activity of chemokines has been discussed in reports known to those skilled in the art (Bruce, L. et al., "Radiolabeled Chemokine binding assays," Methods in Molecular Biology (2000) vol. 138, pp129-134; Raphaele, B. et al. "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp143-148; Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp113-120 Humana Press. Totowa, New Jersey).

The biological activities of CXCL10 include, for example, binding to the CXCL10 receptor (CXCR3), CXCL10-induced calcium flux, CXCL10-induced cell chemotaxis, CXCL10 binding to glycosaminoglycans, and CXCL10 oligomerization.
Methods for measuring the biological activity of CXCL10 include a method for measuring the cell migration activity of CXCL10, the Reporter assay using a cell line that stably expresses CXCR3 (see PLoS One. 2010 Sep 13;5(9):e12700.), and the PathHunter ^TM β-Arrestin recruitment assay, which utilizes B-Arrestin recruitment, which is induced early in GPCR signaling.

Interleukin 12 (IL-12) is a heterodimeric cytokine consisting of disulfide-linked glycosylated polypeptide chains of 30 and 40 kD. The cytokine is synthesized and secreted by antigen-presenting cells, including dendritic cells, monocytes, macrophages, B cells, Langerhans cells and keratinocytes, as well as natural killer (NK) cells. IL-12 mediates a variety of biological processes and has been referred to as NK cell stimulating factor (NKSF), T cell stimulating factor, cytotoxic T lymphocyte maturation factor, and EBV-transformed B cell lineage factor.

Interleukin-12 binds to IL-12 receptors expressed on the plasma membrane of cells (e.g., T cells, NK cells) and can thereby alter (e.g., initiate, block) biological processes. For example, binding of IL-12 to the IL-12 receptor stimulates proliferation of preactivated T cells and NK cells, enhances the cytolytic activity of cytotoxic T cells (CTL), NK cells and LAK (lymphokine-activated killer) cells, induces the production of gamma interferon (IFNγ) by T cells and NK cells, and induces differentiation of naive Th0 cells into Th1 cells that produce IFNγ and IL-2. In particular, IL-12 is absolutely necessary for the generation of cytolytic cells (e.g., NK, CTL) and for initiating cellular immune responses (e.g., Th1 cell-mediated immune responses). Thus, IL-12 is absolutely critical in the generation and regulation of both protective immunity (e.g., eradication of infectious diseases) and pathological immune responses (e.g., autoimmunity).

Methods for measuring the biological activity of IL-12 include measuring the cell proliferation activity of IL-12, STAT4 reporter assay, cell activation by IL-12 (cell surface marker expression, cytokine production, etc.), and promotion of cell differentiation by IL-12.

The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The first members of the family, CD28 and ICOS, were discovered by their functional effects on increasing T cell proliferation after addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260). PD-1 was discovered by screening for differential expression in apoptotic cells (Ishida et al. (1992) EMBO J. 11:3887-95). The other members of the family, CTLA-4 and BTLA, were discovered by screening for differential expression in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 all have unpaired cysteine residues that allow for homodimerization. In contrast, PD-1 is thought to exist as a monomer and does not have the unpaired cysteine characteristic of other CD28 family members.

The PD-1 gene encodes a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily. PD-1 contains a membrane-proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM). PD-1 is structurally similar to CTLA-4 but lacks the MYPPPY motif (SEQ ID NO:102) that is important for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified, and it has been shown that binding to PD-1 negatively regulates T cell activation (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not other CD28 family members. PD-L1, one of the ligands for PD-1, is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9). PD-1 and PD-L1 interaction results in a reduction in tumor-infiltrating lymphocytes, decreased T cell receptor-mediated proliferation, and immune evasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-2 with PD-L2 is similarly inhibited (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

PD-1 is an inhibitory member of the CD28 family expressed on activated B cells, T cells, and myeloid cells. PD-1-deficient animals develop a variety of autoimmune phenotypes, including autoimmune cardiomyopathy and a lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 has been shown to play an important role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunia and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 13:510). In mouse B cell tumor lines, the ITSM of PD-1 was found to be essential in inhibiting BCR-mediated ^Ca2+ flux and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

In some embodiments of the invention, the ligand is a cytokine.
Cytokines are a family of secreted cell signaling proteins involved in immunoregulatory and inflammatory processes, which are secreted by glial cells of the nervous system and by many cells of the immune system. Cytokines can be classified as proteins, peptides or glycoproteins, and encompass a large and diverse family of regulators. Cytokines bind to cell surface receptors and induce intracellular signaling, which can result in the regulation of enzyme activity, up- or down-regulation of some genes and their transcription factors, or feedback inhibition, etc.
In some embodiments, the cytokines of the present invention include immune regulators such as interleukins (IL) and interferons (IFN). Suitable cytokines can include proteins from one or more of the following types: the four α-helical bundle family (which includes the IL-2 subfamily, the IFN subfamily and the IL-10 subfamily); the IL-1 family (which includes IL-1 and IL-8), and the IL-17 family. Cytokines can also include those classified as type 1 cytokines (e.g., IFN-γ, TGF-β, etc.), which enhance cellular immune responses, or type 2 cytokines (e.g., IL-4, IL-10, IL-13, etc.), which favor antibody responses.

In some embodiments of the invention, the ligand is a chemokine. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be beneficial to express certain chemokine genes, for example in conjunction with cytokine genes, in order to recruit other immune system components to the treatment site. Such chemokines include CXCL10, RANTES, MCAF, MIP1-α, and MIP1-β. Those skilled in the art will recognize that certain cytokines are also known to have chemoattractant properties and may be classified under the term chemokine.

In some embodiments of the present invention, modified forms of cytokines, chemokines, etc. (e.g., Annu Rev Immunol. 2015;33:139-67.) or fusion proteins containing them (e.g., Stem Cells Transl Med. 2015 Jan;4(1):66-73.) can be used as ligands.

In some embodiments of the present invention, the ligand is selected from CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. The CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra may have the same sequence as naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra, or may be a variant that has a different sequence from naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra but retains the physiological activity of the corresponding natural ligand. To obtain a modified ligand, the ligand sequence may be artificially modified for various purposes, and preferably, a modified ligand is obtained by adding a modification that is not susceptible to protease cleavage (protease resistant).

Examples of Ligand-Binding Molecules In some embodiments of the present invention, the ligand-binding molecule comprises only a single domain antibody into which a protease cleavage sequence has been introduced. In some other embodiments of the present invention, the ligand-binding molecule comprises an Fc region. When an Fc region of an IgG antibody is used, the type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region comprising one of the amino acid sequences shown in SEQ ID NOs: 103, 104, 105, and 106, or an Fc region mutant obtained by modifying these Fc regions, can be used. In some embodiments of the present invention, the ligand-binding molecule comprises an antibody constant region.

In some embodiments of the present invention, the ligand-binding molecule is a fusion protein of a single domain antibody and an Fc region into which a protease cleavage sequence has been introduced. In a more specific embodiment, the ligand-binding molecule comprises, from the N-terminus to the C-terminus, a series of peptide chains consisting of a single domain antibody into which a protease cleavage sequence has been introduced-antibody Fc region. In another specific embodiment, the ligand-binding molecule is a dimeric protein comprising two series of peptide chains consisting of a single domain antibody into which a protease cleavage sequence has been introduced-antibody hinge region-antibody Fc region. In another specific embodiment, the ligand-binding molecule is a dimeric protein comprising two series of peptide chains consisting of a single domain antibody into which a protease cleavage sequence has been introduced-antibody hinge region-antibody CH2-antibody CH3.

In some embodiments of the present invention, the uncleaved ligand-binding molecule forms a complex with the ligand through antigen-antibody binding. In more specific embodiments, the complex between the ligand-binding molecule and the ligand is formed by a non-covalent bond between the ligand-binding molecule and the ligand, such as an antigen-antibody bond.
Therefore, the present invention also provides a method for producing a complex, which comprises the steps of contacting a ligand-binding molecule with a ligand and recovering a complex formed by binding the ligand to the ligand-binding molecule. In the present invention, the ligand-binding molecule and the ligand can be contacted under conditions that allow binding between the two. The obtained complex can be made into a pharmaceutical composition, for example, by blending with a pharma- ceutically acceptable carrier.

Fusion protein in which a ligand and a ligand-binding molecule are fused In some embodiments of the present invention, an uncleaved ligand-binding molecule is fused to a ligand to form a fusion protein, and the ligand-binding molecule portion and the ligand portion in the fusion protein further interact with each other through antigen-antibody binding. The ligand-binding molecule and the ligand can be fused via a linker or without a linker. Even if the ligand-binding molecule and the ligand in the fusion protein are fused via a linker or without a linker, the non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. In other words, even in an embodiment in which the ligand-binding molecule is fused to the ligand, the non-covalent bond between the ligand-binding molecule portion and the ligand portion is similar to that in an embodiment in which the ligand-binding molecule and the ligand are not fused. When the ligand-binding molecule is cleaved, the non-covalent bond is weakened. That is, the bond between the ligand-binding molecule and the ligand is weakened.
In a preferred embodiment of the present invention, the ligand-binding molecule and the ligand are fused via a linker. The linker used when fusing the ligand-binding molecule and the ligand may be any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996), but in this embodiment, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. Examples of peptide linkers include, but are not limited to, the following:
Ser
Gly-Ser (GS)
Ser-Gly (SG)
Gly-Gly-Ser (GGS)
Gly-Ser-Gly (GSG)
Ser-Gly-Gly (SGG)
Gly-Ser-Ser (GSS)
Ser-Ser-Gly (SSG)
Ser-Gly-Ser (SGS)
Gly.Gly.Gly.Ser (GGGS, SEQ ID NO:83)
Gly.Gly.Ser.Gly (GGSG, SEQ ID NO: 84)
Gly.Ser.Gly.Gly (GSGG, SEQ ID NO: 85)
Ser.Gly.Gly.Gly (SGGG, SEQ ID NO: 86)
Gly.Ser.Ser.Gly (GSSG, SEQ ID NO:87)
Gly.Gly.Gly.Gly.Gly.Ser (GGGGS, SEQ ID NO:88)
Gly.Gly.Gly.Ser.Gly (GGGSG, SEQ ID NO: 89)
Gly.Gly.Ser.Gly.Gly (GGSGG, SEQ ID NO:90)
Gly.Ser.Gly.Gly.Gly.Gly (GSGGG, SEQ ID NO:91)
Gly.Ser.Gly.Gly.Ser (GSGGS, SEQ ID NO:92)
Ser.Gly.Gly.Gly.Gly.Gly (SGGGG, SEQ ID NO:93)
Gly.Ser.Ser.Gly.Gly (GSSGG, SEQ ID NO:94)
Gly.Ser.Gly.Ser.Gly.Gly (GSGSG, SEQ ID NO:95)
Ser.Gly.Gly.Ser.Gly (SGGSG, SEQ ID NO:96)
Gly.Ser.Ser.Ser.Gly (GSSSG, SEQ ID NO:97)
Gly.Gly.Gly.Gly.Gly.Gly.Gly.Ser (GGGGGS, SEQ ID NO:98)
Ser Gly Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 99)
Gly.Gly.Gly.Gly.Gly.Gly.Gly.Gly.Ser (GGGGGGS, SEQ ID NO:100)
Ser Gly Gly Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 101)
(Gly.Gly.Gly.Gly.Gly.Ser (GGGGS, SEQ ID NO:88)
(Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 93)
[n is an integer of 1 or more], etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

Synthetic chemical linkers (chemical crosslinkers) are crosslinkers commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), etc., which are commercially available.

In some embodiments of the present invention, the fusion protein of a ligand-binding molecule and a ligand is a fusion protein in which the N-terminus of the ligand-binding molecule and the C-terminus of the ligand are fused with or without a linker.
Some embodiments of the structure of the fusion protein of the ligand-binding molecule and ligand of the present invention are listed below, in order from the N-terminus to the C-terminus.
Ligand - Single Domain Antibody Ligand - Linker - Single Domain Antibody Ligand - Single Domain Antibody - Antibody Constant Region (or Fragment)
Ligand - Linker - Single domain antibody - Antibody constant region (or fragment)
Ligands - Single Domain Antibodies - Antibody Hinge Regions - Antibody CH2 - Antibody CH3
Ligand - Linker - Single Domain Antibody - Antibody Hinge Region - Antibody CH2 - Antibody CH3

Treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) refers to a clinical intervention intended to modify the natural course of the individual being treated, and may be performed for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the single domain antibody-containing ligand binding molecules of the present invention can control the biological activity of the ligand and are used to delay the onset of the disease or slow the progression of the disease.

Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions (drugs) comprising the single domain antibody-containing ligand binding molecule and a pharma- ceutical carrier, pharmaceutical compositions (drugs) comprising the single domain antibody-containing ligand binding molecule, a ligand and a pharma- ceutical carrier, and pharmaceutical compositions (drugs) comprising a fusion protein in which the single domain antibody-containing ligand binding molecule and a ligand are fused together and a pharma- ceutical carrier.

In the present invention, the pharmaceutical composition generally refers to a drug for treating or preventing a disease, or for testing or diagnosing a disease.
In the present invention, the term "pharmaceutical composition comprising a ligand-binding molecule" can be rephrased as "a method for treating a disease comprising administering a ligand-binding molecule to a subject" or as "use of a ligand-binding molecule in the manufacture of a medicament for treating a disease". The term "pharmaceutical composition comprising a ligand-binding molecule" can be rephrased as "use of a ligand-binding molecule for treating a disease".
The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can be rephrased as "a method for treating a disease comprising administering the ligand-binding molecule and the ligand to a subject" or as "use of the ligand-binding molecule and the ligand in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can be rephrased as "use of the ligand-binding molecule and the ligand for treating a disease."
The term "pharmaceutical composition comprising a fusion protein" can be rephrased as "a method for treating a disease comprising administering the fusion protein to a subject" or as "use of the fusion protein in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a fusion protein" can be rephrased as "use of the fusion protein for treating a disease."

The pharmaceutical composition of the present invention may be formulated using methods known to those skilled in the art. For example, it may be used parenterally in the form of a sterile solution or suspension injection in water or other pharma- ceutically acceptable liquid. For example, it may be formulated by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dose form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is set so as to obtain an appropriate dose within the indicated range.

Sterile compositions for injection can be formulated according to standard pharmaceutical practice using a vehicle such as distilled water for injection. Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary agents (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80(TM), HCO-50, etc.), can be used in combination.

Oily liquids include sesame oil and soybean oil, and may also be used in combination with benzyl benzoate and/or benzyl alcohol as a solubilizing agent. They may also be combined with buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into a suitable ampule.

The pharmaceutical composition of the present invention is preferably administered parenterally. For example, the composition may be administered in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, the composition may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

The administration method may be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the ligand-binding molecule may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dosage may be set to 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these numerical values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and administration method taking these conditions into consideration.

In some embodiments of the present invention, a composition containing a ligand-binding molecule can be administered to an individual. The ligand-binding molecule administered to an individual binds to a ligand originally present in the individual in, for example, blood, tissue, etc., and is transported further in the body while still bound to the ligand. The ligand-binding molecule transported to the target tissue is cleaved in the target tissue, weakening its binding to the ligand and releasing the ligand bound to the target tissue. The released ligand exerts biological activity in the target tissue and can treat a disease caused by the target tissue. In an embodiment in which the ligand-binding molecule suppresses the biological activity of the ligand when bound to the ligand and the ligand-binding molecule is cleaved specifically in the target tissue, the biological activity of the ligand is not exerted during transport, but is only exerted when cleaved in the target tissue, allowing the disease to be treated and systemic side effects to be suppressed.

The above aspects provide a method for suppressing or inhibiting the systemic action of an endogenous ligand, comprising the step of administering a ligand-binding molecule of the present invention. Alternatively, the present invention provides a pharmaceutical composition for suppressing or inhibiting the systemic action of an endogenous ligand, comprising a ligand-binding molecule as an active ingredient. Alternatively, the present invention relates to the use of a ligand-binding molecule in suppressing or inhibiting the systemic action of an endogenous ligand. Furthermore, the present invention relates to the use of a ligand-binding molecule in the manufacture of a pharmaceutical composition for suppressing or inhibiting the systemic action of an endogenous ligand. In these aspects, the systemic action of an endogenous ligand is first suppressed or inhibited by the ligand-binding molecule of the present invention, and then expressed as a tissue-specific action in the target tissue.

In some embodiments of the present invention, a composition containing a ligand-binding molecule and a composition containing a ligand can be administered to an individual separately or simultaneously. A composition containing both a ligand-binding molecule and a ligand can also be administered to an individual. When a composition containing both a ligand-binding molecule and a ligand is administered to an individual, the ligand-binding molecule and the ligand in the composition may form a complex. When both a ligand-binding molecule and a ligand are administered to an individual, the ligand-binding molecule binds to the ligand administered to the individual and is transported in the body while still bound to the ligand. The ligand-binding molecule transported to the target tissue is cleaved in the target tissue, the binding to the ligand is weakened, and the ligand bound to the target tissue can be released. The released ligand exerts biological activity in the target tissue and can treat a disease caused by the target tissue. In an embodiment in which the ligand-binding molecule suppresses the biological activity of the ligand when bound to the ligand and the ligand-binding molecule is cleaved specifically to the target tissue, the biological activity of the ligand is not exerted during transport, but is only exerted when cleaved in the target tissue, thereby treating a disease and suppressing systemic side effects. A ligand-binding molecule administered to an individual can bind not only to the ligand administered to the individual, but also to ligands that are originally present in the individual, and can transport the ligand originally present in the individual or the ligand administered to the individual in the bound state within the body.
That is, the present invention also provides a method for producing a ligand-ligand-binding molecule complex, which comprises the steps of contacting a ligand-binding single domain antibody-containing molecule with a ligand and recovering a complex consisting of the single domain antibody-containing molecule and the ligand. The complex of the present invention can be made into a pharmaceutical composition, for example, by blending it with a pharma- ceutical acceptable carrier.

In some embodiments of the present invention, a fusion protein in which a ligand-binding molecule and a ligand are fused can be administered to an individual. In some of these embodiments, the ligand-binding molecule and the ligand in the fusion protein form a fusion protein with or without a linker, but a non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. When a fusion protein in which a ligand-binding molecule and a ligand are fused is administered to an individual, the fusion protein is transported in the body, and the ligand-binding molecule portion in the fusion protein is cleaved in the target tissue, thereby weakening the non-covalent bond of the ligand-binding molecule portion to the ligand, and a portion of the ligand and the ligand-binding molecule are released from the fusion protein. The released ligand and a portion of the ligand-binding molecule exert the biological activity of the ligand in the target tissue, and can treat a disease caused by the target tissue. In an embodiment in which the ligand-binding molecule suppresses the biological activity of the ligand when bound to the ligand and the ligand-binding molecule is cleaved specifically to the target tissue, the biological activity of the ligand in the fusion protein during transport is not exerted, but is only exerted when cleaved in the target tissue, and the disease can be treated, and systemic side effects can be suppressed.

The above aspects provide a method for delivering a ligand. That is, the present invention provides a method for delivering a ligand to a target tissue, comprising the step of administering a ligand bound to a ligand conjugate of the present invention. Alternatively, the present invention provides a composition for delivering a ligand to a target tissue, comprising a ligand bound to a ligand conjugate. Furthermore, the present invention relates to the use of a ligand bound to a ligand conjugate in delivering a ligand to a target tissue. Furthermore, the present invention relates to the use of a ligand bound to a ligand conjugate in the manufacture of a composition for delivering a ligand to a target tissue.

The present invention also relates to a method for producing a ligand-binding molecule containing a single domain antibody whose binding to a ligand is weakened in a cleaved state, or a fusion protein in which the ligand-binding molecule is fused with a ligand. In one embodiment of the present invention, there is provided a method for producing a ligand-binding molecule or a fusion protein, which comprises introducing a protease cleavage sequence into a single domain antibody contained in the ligand-binding molecule.

Examples of methods for introducing a protease cleavage sequence into a single domain antibody contained in a ligand-binding molecule include a method of inserting a protease cleavage sequence into the amino acid sequence of a single domain antibody contained in a ligand-binding molecule, and a method of replacing part of the amino acid sequence of a single domain antibody contained in a ligand-binding molecule with a protease cleavage sequence.

"Inserting" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts without deleting it, and connecting the two parts with amino acid sequence A (i.e., creating a new amino acid sequence such as "first half of amino acid sequence B-amino acid sequence A-second half of amino acid sequence B"). "Introducing" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts and connecting the two parts with amino acid sequence A. In addition to "inserting" amino acid sequence A into amino acid sequence B, it is also possible to delete one or more amino acid residues, including those in amino acid sequence B adjacent to amino acid sequence A, and then connect the two parts with amino acid sequence A (i.e., replacing part of amino acid sequence B with amino acid sequence A).

An example of a method for obtaining a single domain antibody-containing ligand-binding molecule is a method for obtaining a single domain antibody capable of binding to a ligand. The single domain antibody can be obtained, for example, by a method using a known method for producing a single domain antibody.
The single domain antibody obtained by this production method may be used as it is as a ligand-binding molecule, or the obtained single domain antibody may be linked to another amino acid sequence for use.

Methods for producing single domain antibodies are similar to those for producing conventional antibodies and are well known to those skilled in the art. For example, in the case of monoclonal antibodies, they may be produced by the hybridoma method (Kohler and Milstein, Nature 256:495 (1975)) or recombinant method (U.S. Patent No. 4,816,567). They may also be isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)). They may also be isolated from single B cell clones (N. Biotechnol. 28(5): 253-457 (2011)).

The ligand-binding molecule of the present invention can be produced by introducing a protease cleavage sequence into the ligand-binding molecule. More specifically, the ligand-binding molecule of the present invention can be produced by introducing a protease cleavage sequence into a single domain antibody in the ligand-binding molecule. Optionally, it can be confirmed whether the ligand-binding molecule is cleaved by treatment with a protease that corresponds to the protease cleavage sequence. For example, it can be confirmed whether the protease cleavage sequence has been cleaved by contacting a ligand-binding molecule into which a protease cleavage sequence has been introduced with a protease and confirming the molecular weight of the product after protease treatment by electrophoresis such as SDS-PAGE.

The present invention also relates to polynucleotides encoding single domain antibody-containing ligand binding molecules that exhibit reduced binding to a ligand in a cleaved state, or polynucleotides encoding fusion proteins in which the ligand binding molecule is fused to a ligand.

The polynucleotide of the present invention is usually carried (inserted) in a suitable vector and introduced into a host cell. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if E. coli is used as the host, a cloning vector such as pBluescript vector (Stratagene) is preferred, but various commercially available vectors can be used. When a vector is used for the purpose of producing the ligand-binding molecule or fusion protein of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it expresses the ligand-binding molecule in a test tube, in E. coli, in cultured cells, or in an individual organism. For example, the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472(1988)) for individual organisms are preferred. The DNA of the present invention can be inserted into a vector by standard methods, for example, a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

There are no particular limitations on the host cells, and various host cells can be used depending on the purpose. Examples of cells for expressing ligand-binding molecules or fusion proteins include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vectors can be introduced into host cells by known methods, such as calcium phosphate precipitation, electric pulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 9.1-9.9), lipofectamine method (GIBCO-BRL), and microinjection.

Appropriate secretion signals can be incorporated into the ligand-binding molecule or fusion protein of interest to secrete the ligand-binding molecule or fusion protein expressed in a host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment. These signals can be endogenous to the ligand-binding molecule or fusion protein of interest or can be heterologous signals.

The method for producing a ligand-binding molecule or fusion protein typically includes a step of recovering the ligand-binding molecule or fusion protein. When the ligand-binding molecule or fusion protein of the present invention is secreted into the culture medium, the culture medium is recovered to recover the ligand-binding molecule or fusion protein in the above production method. When the ligand-binding molecule or fusion protein of the present invention is produced intracellularly, the cells are first lysed, and then the ligand-binding molecule or fusion protein is recovered.

Known methods can be used to recover and purify the ligand binding molecules or fusion proteins of the invention from recombinant cell cultures, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.

It will be understood by those skilled in the art that any combination of one or more of the embodiments described in this specification is included in the present invention, so long as it is not technically inconsistent based on the technical common knowledge of those skilled in the art. In addition, an invention that excludes any combination of one or more of the embodiments described in this specification from the present invention is also to be considered as an invention intended and described in this specification, so long as it is not technically inconsistent based on the technical common knowledge of those skilled in the art.

The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced in light of the general description above.

Example 1 Issues with previously reported immunocytokines and protease-activated cytokines Immunocytokines targeting antigens expressed in cancer tissues have generally been produced by fusing the target cytokine to the end of the targeting IgG or scFv (Expert Opin Investig Drugs. 2009 Jul;18(7):991-1000., Curr Opin Immunol. 2016 Jun;40:96-102.). Since cytokines such as IL-2, IL-12, and TNF are highly toxic, it is expected that these cytokines can be delivered to the cancer local area by antibodies to work locally in the cancer, thereby reducing side effects and enhancing the effect (Non-Patent Documents 4, 5, 6). However, all of these have issues such as insufficient clinical efficacy when administered systemically, narrow therapeutic window, and high toxicity that prevents systemic administration. The main reason for this is that even if it is an immunocytokine, if it is administered systemically, it is exposed to the entire body, and therefore may act systemically and exhibit toxicity, or it can only be administered in extremely low doses to avoid toxicity. In addition, since immunocytokines that bind to cancer antigens are internalized by cancer cells in the tumor and disappear, in some cases it becomes difficult to expose the cytokine to the tumor site. There is also a report that the antitumor effect was the same for an immunocytokine in which IL-2 was fused to an antibody that binds to a cancer antigen and an immunocytokine in which IL-2 was fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

As a method to reduce the systemic effect, which is a major issue for immunocytokines, molecules have been reported in which the cytokine and cytokine receptor are linked by a linker that is cleaved by a protease that is highly expressed in cancer. Cytokines are inhibited by the cytokine receptor linked by the linker, but when the linker is cleaved by a protease, the cytokine receptor is released and the cytokine becomes active. Examples include a molecule in which TNFalpha and TNFR are linked by a linker that is cleaved by uPA (Cancer Immunol Immunother. 2006 Dec;55(12):1590-600.), and a molecule in which IL-2 and IL-2R are linked by a linker that is cleaved by MMP-2 (Immunology. 2011 Jun;133(2):206-20). However, in these molecules, the cytokines have biological activity even before the linker is cleaved, and cleavage of the linker only increases activity by about 10 times.
This is due to two reasons: first, the affinity between the cytokine and the cytokine receptor is not strong, so that the cytokine retains a certain degree of activity even before protease cleavage; second, the cytokine receptor can bind to the cytokine even after the linker is cleaved by the protease, thereby inhibiting the biological activity of the cytokine.

Example 2: Issues in the application of chemokines to cancer immunotherapy Chemokines (Nature Immunology 9, 949 - 952 (2008)) are basic proteins that exert their effects via G protein-coupled receptors, and are a group of cytokines. They act on specific leukocytes that express the receptor, and have the activity of causing the leukocytes to migrate in the direction of the substance's concentration gradient (chemotaxis) (Nat Cell Biol. 2016 Jan;18(1):43-53.). Chemokines are produced in large quantities at sites of inflammation, and are known to cause the migration of leukocytes from within blood vessels into inflamed tissues.
Since chemokines can control the migration of white blood cells, they can be used in cancer immunotherapy. If T cells, antigen-presenting cells, M1 macrophages, etc. can be induced to migrate to the local area of solid cancer, it is thought that an antitumor effect can be induced. Cytokines can also exert their effects through systemic administration, but chemokines induce cell migration to tissues with high concentrations due to a concentration gradient, so the expected effect cannot be obtained by systemic administration of chemokines. Therefore, cancer immunotherapy (chemokine therapy) using systemic administration of chemokines is thought to be unrealistic.

Example 3: Concept of a ligand-binding molecule capable of releasing a target tissue-specific ligand by introducing a protease cleavage sequence. As shown in Examples 1 and 2, previously reported cytokine/chemokine therapies have the following problems.
1. In the case of immunocytokines, even if cytokines are targeted to solid tumors using antibodies, they act throughout the body, causing side effects, or they can only be administered in low doses to avoid side effects, meaning that high exposure cannot be achieved in the tumor.
2. In the case of a cytokine that is activated by a protease, in which a cytokine receptor (or antibody) and the cytokine are connected by a linker that can be cleaved by a protease, neutralization of the cytokine activity is insufficient and the cytokine retains a certain degree of activity even before protease cleavage.
3. In the case of cytokines that are activated by proteases, the cytokine receptor (or antibody) can bind to the cytokine even after the linker is cleaved by the protease, thereby inhibiting the biological activity of the cytokine.

In order to solve this problem, it was considered important to meet the following conditions:
1. In the whole body, ligands such as cytokines or chemokines are sufficiently inhibited (biological activity is minimized) by ligand-binding molecules.
2. The biological activity of the ligand is restored by cleavage with a protease (becoming an active ligand).
3. The ligand-binding molecule loses its ligand-binding activity upon cleavage by a protease.

As a pharmaceutical composition that satisfies the above conditions, we have devised a molecule whose binding to a ligand is weakened by cleavage of the cleavage site. First, a single domain antibody against a ligand is obtained, and then a cleavage site is introduced into the single domain antibody, allowing the production of a ligand-binding molecule that contains the single domain antibody containing the cleavage site.

Example 4 Examples of Ligand-Binding Molecules Containing Single Domain Antibodies with an Introduced Cleavage Site FIGS. 1 to 4 show examples of ligand-binding molecules containing single domain antibodies with an introduced cleavage site .
FIG. 1 shows an example of a ligand-binding molecule containing only a single domain antibody into which a cleavage site has been introduced. When the cleavage site is not cleaved, the single domain antibody can bind to the ligand, and if the affinity of the single domain antibody for the ligand is sufficiently strong, the biological activity of the ligand is sufficiently inhibited. Even if this ligand-binding molecule and the ligand are administered systemically, the ligand in the complex of the ligand-binding molecule and the ligand is neutralized and does not exhibit its biological activity, and the complex of the ligand-ligand-binding molecule has a longer half-life than the ligand alone. In the case where the cleavage site is a protease cleavage sequence that is cleaved by a tumor tissue-specific protease, when the protease cleavage sequence in the single domain antibody is cleaved by a protease highly expressed in tumor tissue, the complex formed by the systemically administered ligand-binding molecule and the ligand is no longer able to bind to the ligand, the neutralization of the ligand is released, and the single domain antibody can exhibit a biological effect in the tumor tissue.

Figure 2 shows an example of a fusion polypeptide in which a ligand is fused to a ligand-binding molecule containing only a single domain antibody with an introduced cleavage site. When the cleavage site is not cleaved, the single domain antibody in the fusion polypeptide can bind to the ligand, and if the affinity of the single domain antibody for the ligand is sufficiently strong, the biological activity of the ligand is sufficiently inhibited. Even if this fusion polypeptide is administered systemically, the ligand in the fusion polypeptide is neutralized and does not exert its biological activity, and the fusion polypeptide has a longer half-life than the ligand alone. In the case where the cleavage site is a protease cleavage sequence that is cleaved by a tumor tissue-specific protease, when the protease cleavage sequence in the single domain antibody is cleaved by a protease highly expressed in tumor tissue, the single domain antibody in the fusion polypeptide cannot bind to the ligand, the neutralization of the ligand is released, and a part of the fusion polypeptide containing the ligand is released, which can exert a biological effect in the tumor tissue.

Figures 3 and 4 show examples of a single domain antibody into which a cleavage site has been introduced, a ligand-binding molecule containing an antibody hinge region and an antibody Fc region, and a fusion polypeptide of the ligand-binding molecule and the ligand. As in the embodiments of Figures 1 and 2, the ligand can be neutralized by the single domain antibody in the uncleaved state, and the ligand can be released in the cleaved state to exert a biological effect. In addition, in the examples of Figures 3 and 4, since the complex or fusion polypeptide in the uncleaved state contains an antibody Fc region, it is expected that the half-life in the uncleaved state will be even longer than in the examples of Figures 1 and 2.

Example 5 Anti-human IL-6R single domain antibody-containing ligand-binding molecule having a protease cleavage sequence introduced therein
5-1 Preparation of ligand-binding molecule by introduction of protease cleavage sequence into polypeptide containing anti-human IL-6R single domain antibody A protease cleavage sequence was inserted into the single domain antibody in the polypeptide IL6R90-G1T3dCHdC (SEQ ID NO: 122) in which the C-terminus of a single domain antibody against human IL-6R (SEQ ID NO: 120) is fused with the antibody hinge region and the N-terminus of the antibody Fc region sequence shown in SEQ ID NO: 121, to prepare a single domain antibody-containing ligand-binding molecule containing a protease cleavage sequence. First, a sequence containing a peptide sequence (LSGRSDNH, SEQ ID NO: 3) that has been reported to be cleaved by matriptase (MT-SP1) expressed specifically in cancer was inserted into the polypeptide IL6R90-G1T3dCHdC to prepare the ligand-binding molecule shown in Table 2. The molecule was expressed by transient expression using Expi293 (Life Technologies) and purified by a method known to those skilled in the art using protein A. As a control molecule that does not contain a protease cleavage sequence, IL6R90-G1T3dCHdC was expressed and purified. The ligand-binding molecules and control molecules produced were dimeric proteins as shown in FIG.

5-2. Evaluation of binding to human IL-6R of anti-human IL-6R single domain antibody-containing ligand binding molecules incorporating a protease cleavage sequence
5-2-1 Protease treatment Recombinant Human Matriptase/ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) was added to 0.1 mg/mL of the ligand-binding molecule prepared in Example 5-1 to a final concentration of 25 nM, and the mixture was incubated at 37°C overnight to prepare a solution containing protease-treated ligand-binding molecules. A solution containing protease-untreated ligand-binding molecules was prepared by adding the same volume of PBS instead of protease and storing under the same conditions.

5-2-2 Confirmation of protease cleavage of ligand-binding molecules (SDS-PAGE)
Whether the ligand-binding molecule was cleaved by the protease treatment performed in Example 5-2-1 was confirmed by SDS-PAGE. 9 μL of the solution containing the protease-treated ligand-binding molecule/protease-untreated ligand-binding molecule prepared in Example 5-2-1 was mixed with 3 μL of sample buffer and incubated at 95°C for 1 minute. Next, electrophoresis was performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the protein was stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 5. As shown in Figure 5, the control molecules not containing a protease cleavage sequence (lanes 12 and 13) had bands at the same position regardless of whether they were treated with or not with a protease, whereas the ligand-binding molecules into which each protease cleavage sequence had been introduced had new bands that appeared only after protease treatment, indicating that the single-domain antibody-containing ligand-binding molecules into which each protease cleavage sequence had been introduced were cleaved by protease treatment.

5-2-3 Evaluation of binding of protease-treated ligand-binding molecules/protease-untreated ligand-binding molecules to human IL-6R 60 μL of PBS was added to 20 μL of the solution containing the protease-treated ligand-binding molecules/protease-untreated ligand-binding molecules prepared in Example 5-2-1 to prepare an IL-6R binding evaluation sample. IL-6R binding evaluation of the sample was measured by biolayer interferometry (BLI method). The IL-6R binding evaluation sample and IL-6R were dispensed into a tilted bottom (TW384) Microplate (Forte bio, 18-5076). A Protein G sensor (ForteBio, 18-0022) was hydrated with PBS, and measurements were performed with Octet RED 384 at a setting of 25 degrees. A baseline measurement was performed for 30 seconds in the wells containing PBS, and then the antibody was allowed to bind to the Protein G sensor for 200 seconds. Again, a baseline measurement was performed for 30 seconds in wells containing PBS, followed by measurement of binding for 180 seconds in wells containing 500 nM human IL-6R, and measurement of dissociation for 180 seconds in wells containing PBS. A real-time binding graph showing the state of binding is shown in Figure 6. As shown in Figure 6, when each ligand-binding molecule containing a protease cleavage sequence was used, the amount of human IL-6R binding was reduced compared to when a ligand-binding molecule not treated with protease was used.

Example 6: Preparation and evaluation of a fusion protein of an anti-human IL-6R single domain antibody-containing ligand binding molecule containing a protease cleavage sequence and human IL-6R (ligand binding molecule-IL-6R fusion protein)
6-1. Preparation of fusion proteins of anti-human IL-6R single domain antibody-containing ligand-binding molecule with a protease cleavage sequence introduced therein and human IL-6R The N-terminus of the ligand-binding molecule prepared in Example 5-1 was fused to the C-terminus of human IL-6R (SEQ ID NO: 128) via various linkers consisting of glycine and serine to prepare ligand-binding molecule-IL-6R fusion proteins (Table 3). These fusion proteins were expressed by transient expression using Expi293 (Life Technologies) by a method known to those skilled in the art, and purified by a method known to those skilled in the art using protein A. Each of the fusion proteins prepared is a dimeric protein having two peptide chains with the sequences shown in Table 3.

As a control molecule not containing a protease cleavage sequence, a molecule (Table 4) was prepared in which the N-terminus of IL6R90-G1T3dCHdC (SEQ ID NO: 122) was fused to the C-terminus of IL-6R via a linker, and was expressed and purified. Each of the control molecules prepared is also a dimeric protein having two peptide chains with the sequences shown in Table 4.

6-2 Evaluation of protease cleavage of ligand-binding molecule-IL-6R fusion protein
6-2-1 Protease Treatment Recombinant Human Matriptase/ST14 Catalytic Domain (hMT-SP1, R&D Systems 3946-SE-010) was added to 0.1 mg/mL of the ligand-binding molecule-IL-6R fusion protein prepared in Example 6-1 to a final concentration of 25 nM, and the mixture was incubated at 37°C overnight to prepare a solution containing the protease-treated fusion protein. The solution containing the protease-untreated fusion protein was prepared by adding the same volume of PBS instead of protease and storing under the same conditions.

6-2-2 Protease cleavage of anti-IL-6R single domain antibody-IL-6R fusion protein (SDS-PAGE)
Whether the ligand-binding molecule-IL-6R fusion protein was cleaved by the protease treatment performed in Example 6-2-1 was confirmed by SDS-PAGE. 9 μL of the solution containing the protease-treated fusion protein/protease-untreated fusion protein prepared in Example 6-2-1 was mixed with 3 μL of sample buffer and incubated at 95°C for 1 minute. Next, electrophoresis was performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the protein was stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 7. As shown in Figure 7, the control molecule not containing a protease cleavage sequence had bands at the same position regardless of whether it was treated with or not with protease, whereas each fusion protein containing a single domain antibody-containing ligand-binding molecule into which a protease cleavage sequence had been introduced had a new band that appeared only after protease treatment, indicating that each fusion protein containing a single domain antibody-containing ligand-binding molecule into which a protease cleavage sequence had been introduced was cleaved by protease treatment.

6-2-3 Preparation of biotinylated anti-IL-6R single domain antibody-containing molecule As a method for detecting the release of IL-6R fused to a ligand-binding molecule, a method was used in which biotin was added to a single domain antibody-containing molecule that recognizes IL-6R, and the binding of free IL-6R to the biotinylated anti-IL-6R single domain antibody-containing molecule was detected. The biotinylated anti-IL-6R single domain antibody-containing molecule was prepared as follows. An anti-IL-6R single domain antibody-containing molecule having the same single domain antibody partial sequence as IL6R90-G1T3dCH1dC (SEQ ID NO: 122) was prepared, and a biotin-addition sequence (AviTag sequence, SEQ ID NO: 159) was added to the C-terminus to prepare IL6R90-FcBAPdC (SEQ ID NO: 160). A gene fragment encoding IL6R90-FcBAPdC was prepared and introduced into an animal cell expression vector by a method known to those skilled in the art. At this time, a gene expressing EBNA1 and a gene expressing biotin ligase were simultaneously introduced, and biotin was added for the purpose of biotin labeling. The cells into which the genes were introduced were cultured at 37°C and 8% CO2, and the target biotinylated anti-IL-6R single domain antibody-containing molecule (IL6R90-bio) was secreted into the culture supernatant. IL6R90-bio was purified from the culture supernatant by a method known to those skilled in the art.

6-2-4 Evaluation of IL-6R release from fusion protein by protease treatment IL-6R release by protease treatment of the fusion protein was evaluated by biolayer interferometry (BLI) using the biotinylated anti-IL-6R single domain antibody-containing molecule (IL6R90-bio) prepared in Example 6-2-3.

The protease-treated fusion protein, protease-untreated fusion protein, and IL6R90-bio prepared in Example 6-2-1 were dispensed into different wells of tilted bottom (TW384) Micro plates (ForteBio, 18-5076). Streptavidin biosensors (ForteBio, 18-5021) were hydrated with PBS and measurements were performed with Octet RED 384 at 27°C. A baseline measurement was performed for 30 seconds in the wells containing PBS, after which IL6R90-bio was allowed to bind to the Streptavidin sensor for 200 seconds. A baseline measurement was performed again for 30 seconds in the wells containing PBS, after which binding was measured for 180 seconds in the wells containing the protease-treated fusion protein or the protease-untreated fusion protein, and dissociation was measured for 180 seconds in the wells containing PBS. A real-time binding graph showing the binding is shown in Figure 8. As shown in Figure 8, in the case of a single domain antibody-containing ligand-binding molecule-IL-6R fusion protein into which a protease cleavage sequence had been introduced, the amount of human IL-6R bound to IL6R90-bio increased in the case of protease treatment compared to the case of no protease treatment. In other words, the binding activity of the single domain antibody in the fusion protein to IL-6R was weakened by protease treatment, and IL-6R was released from the fusion protein.

Example 7 Evaluation of introduction of various protease cleavage sequences
7-1 Preparation of IgG antibodies incorporating various protease cleavage sequences An expression vector for MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 119), light chain: MRAL-k0 (SEQ ID NO: 118)), a neutralizing antibody (IgG antibody) against human IL-6R, was prepared by a method known to those skilled in the art.
Table 5 shows peptide sequences known to be cleaved by MMP-2, MMP-7, and MMP-9, as well as peptide sequences containing a flexible linker consisting of a glycine-serine polymer adjacent to these sequences.

These insertion sequences were inserted near the boundary between the heavy chain variable region and constant region of the MRA antibody, resulting in modified heavy chains MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 (SEQ ID NO: 111), MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 (SEQ ID NO: 112), MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 (SEQ ID NO: 113), MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 (SEQ ID NO: 114), MEIVHMP2.1-MEIVHMP2.1G1T4 (SEQ ID NO: 115), MEIVHMP2.3-MEIVHMP2.3G1T4 (SEQ ID NO: 116), and MEIVHMP7.2-MEIVHMP7.2G1T4. (heavy chain SEQ ID NO: 117) was designed, and expression vectors encoding these modified heavy chains were constructed by methods known to those skilled in the art.
These modified heavy chains and MRA light chains were combined to produce the MRA modifications shown in Table 6, which were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art, and purified using Protein A by methods known to those skilled in the art.

7-2. Evaluation of protease cleavage of IgG antibodies containing various protease cleavage sequences
We examined whether the modified MRA prepared in 7-1 could be cleaved by proteases. The proteases used were recombinant human MMP-2 (R&D Systems, 902-MP-010), recombinant human MMP-7 (R&D Systems, 907-MP-010), and recombinant human MMP-9 (R&D Systems, 911-MP-010). The proteases were mixed with 1 mM p-aminophenylmercuric acetate (APMA; abcam, ab112146) and activated at 37°C for 1 and 24 hours, respectively, before use. After incubation for 20 hours at 37°C using 50 nM, 100 nM, or 500 nM protease, 50 μg/mL antibody, and assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer) or 20 mM Tris-HCl, 150 mM NaCl, 5 mM _CaCl2 , pH 7.2 (hereafter referred to as Tris), the cleavage by proteases was evaluated by reducing SDS-PAGE. The results are shown in Figures 11, 12, and 13. The MRA modified antibodies were reacted with the proteases shown in Table 2. In MMP-2, the cleavage was MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4/MRAL-k0, MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4/MRAL-k0, MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4/MRAL-k0, MEIVHMP2.1-MEIVHMP2.1G1T4/MRAL-k0, MEIVHMP2.3-MEIVHMP2.3G1T4/MRAL-k0. In MMP-7, the cleavage was MEIVHMP7.2-MEIVHMP7.2G1T4/MRAL-k0. In MMP-9, the cleavage was MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4/MRAL-k0, Cleavage of MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4/MRAL-k0 was observed.

Example 8: Preparation and evaluation of a fusion protein of an anti-human PD-1 single domain antibody-containing ligand-binding molecule and human PD-1 (ligand-binding molecule-PD-1 fusion protein) incorporating a protease cleavage sequence
8-1. Preparation of fusion proteins of anti-human PD-1 single domain antibody-containing ligand-binding molecule and human PD-1 with a protease cleavage sequence PD102C3-G1T3noCyshinge (SEQ ID NO: 161) and PD102C12-G1T3noCyshinge (SEQ ID NO: 162) were prepared by fusing a human antibody constant region to a single domain antibody that binds to human PD-1 using methods known to those skilled in the art. Next, a protease cleavage sequence was introduced into PD102C3-G1T3noCyshinge and PD102C12-G1T3noCyshinge, and the N-terminus of each was fused to the C-terminus of the extracellular domain of human PD-1 (SEQ ID NO: 163) via various linkers consisting of glycine and serine to prepare ligand-binding molecule-PD-1 fusion proteins (Table 7). These fusion proteins were expressed by transient expression using Expi293 (Life Technologies) using methods known to those skilled in the art, and purified using protein A using methods known to those skilled in the art. Each of the fusion proteins prepared was a dimeric protein having two peptide chains of the sequences shown in Table 7.

8-2. Evaluation of protease cleavage of ligand-binding molecule-PD-1 fusion protein
To the ligand -binding molecule-PD-1 fusion protein (0.1 mg/mL) prepared in Example 8-1, Recombinant Human Matriptase/ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) was added to a final concentration of 25 nM, and the mixture was incubated at 37°C overnight to prepare a solution containing the protease-treated fusion protein. The solution containing the protease-untreated fusion protein was prepared by adding the same volume of PBS instead of protease and storing under the same conditions.

8-2-2. Protease cleavage of anti-PD-1 single domain antibody-PD-1 fusion protein (SDS-PAGE)
Whether the ligand-binding molecule-PD-1 fusion protein was cleaved by the protease treatment performed in Example 8-2-1 was confirmed by SDS-PAGE. 9 μL of the solution containing the protease-treated fusion protein/protease-untreated fusion protein prepared in Example 8-2-1 was mixed with 3 μL of sample buffer and incubated at 95°C for 1 minute. Next, electrophoresis was performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the protein was stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 9. As shown in Figure 9, each fusion protein containing a single domain antibody-containing ligand-binding molecule into which each protease cleavage sequence was introduced had a new band that appeared only after protease treatment, indicating that each fusion protein containing a single domain antibody-containing ligand-binding molecule into which each protease cleavage sequence was introduced was cleaved by protease treatment.

8-2-3. Preparation of biotinylated anti-PD-1 single domain antibody-containing molecule As a method for detecting the release of PD-1 fused to a ligand-binding molecule, a method was used in which biotin was added to a single domain antibody-containing molecule that recognizes PD-1, and the binding of free PD-1 to the biotinylated anti-PD-1 single domain antibody-containing molecule was detected. The biotinylated anti-PD-1 single domain antibody-containing molecule was prepared as follows. A single domain antibody-containing molecule having the same single domain antibody partial sequence as PD102C3-G1T3noCyshinge was prepared, and a biotin addition sequence (AviTag sequence, SEQ ID NO: 159) was added to its C-terminus to prepare PD102C3-FcBAPdC (SEQ ID NO: 168). In addition, an anti-PD-1 single domain antibody-containing molecule having the same single domain antibody partial sequence as PD102C12-G1T3noCyshinge was prepared, and a biotin-addition sequence (AviTag sequence, SEQ ID NO: 159) was added to its C-terminus to prepare PD102C12-FcBAPdC (SEQ ID NO: 169). Gene fragments encoding PD102C3-FcBAPdC and PD102C12-FcBAPdC were prepared and introduced into an animal cell expression vector by a method known to those skilled in the art. At this time, a gene expressing EBNA1 and a gene expressing biotin ligase were simultaneously introduced, and biotin was added for the purpose of biotin labeling. The cells into which the gene was introduced were cultured at 37°C and 8% CO2, and the desired biotinylated anti-PD-1 single domain antibody-containing molecule (PD1-bio) was secreted into the culture supernatant. PD1-bio was purified from the culture supernatant by a method known to those skilled in the art.

8-2-4 Evaluation of PD-1 release from fusion protein by protease treatment PD-1 release by protease treatment of the fusion protein was evaluated by biolayer interferometry (BLI) using the biotinylated anti-PD-1 single domain antibody-containing molecule (PD1-bio) prepared in Example 8-2-3.

The protease-treated fusion protein, the protease-untreated fusion protein, and PD1-bio prepared in Example 8-2-1 were dispensed into different wells of tilted bottom (TW384) Micro plates (ForteBio, 18-5076). PD1-Bio was appropriately selected as PD102C3-FcBAPdC or PD102C12-FcBAPdC so that it would bind to the same epitope as the single domain antibody in the measurement sample. Streptavidin biosensor (ForteBio, 18-5021) was hydrated with PBS, and measurements were performed with Octet RED 384 at 27°C. A baseline measurement was performed for 30 seconds in the well containing PBS, and then PD1-bio was allowed to bind to the Streptavidin sensor for 200 seconds. Again, a baseline measurement was performed for 30 seconds in wells containing PBS, followed by binding measurements for 180 seconds in wells containing either protease-treated or non-protease-treated fusion protein, and dissociation measurements for 180 seconds in wells containing PBS. A real-time binding graph showing the binding is shown in Figure 10. As shown in Figure 10, in the case of a single domain antibody-containing ligand-binding molecule-PD-1 fusion protein containing a protease cleavage sequence, the amount of human PD-1 bound to PD1-bio was increased in the protease-treated case compared to the case of non-protease treatment. In other words, the binding activity of the single domain antibody in the fusion protein to PD-1 was weakened by protease treatment, and PD-1 was released from the fusion protein.

The foregoing invention has been described in detail by way of illustration and illustration for purposes of facilitating a clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

The ligand-binding molecule of the present invention is transported in the body with the ligand still bound, and is cleaved in the diseased tissue, weakening the binding to the ligand, allowing the ligand to be released specifically to the diseased tissue, so that the diseased tissue can be specifically exposed to the ligand. Furthermore, since the ligand-binding molecule suppresses the biological activity of the ligand when transported, there is less risk of the ligand acting systemically, making it extremely useful for treating diseases.

Claims (15)

A ligand-binding molecule, comprising a single domain antibody capable of binding to a ligand and having at least one cleavage site introduced therein, wherein the binding of the ligand-binding molecule to the ligand when the cleavage site is cleaved is weakened compared to the binding of the ligand-binding molecule to the ligand when the cleavage site is not cleaved. The ligand binding molecule of claim 1, wherein the cleavage site comprises a protease cleavage sequence. The ligand binding molecule of claim 2, wherein the protease is a target tissue-specific protease. A ligand-binding molecule, the ligand-binding molecule comprising a single domain antibody, the single domain antibody capable of binding to a ligand and comprising at least one protease cleavage sequence, the protease cleavage sequence being cleavable by a target tissue-specific protease, and the binding of the ligand-binding molecule to the ligand in a state where the protease cleavage sequence is cleaved is weakened compared to the binding of the ligand-binding molecule to the ligand in a state where the cleavage site is not cleaved. The ligand-binding molecule according to any one of claims 2 to 4, wherein the protease is a cancer tissue-specific protease or an inflammatory tissue-specific protease. The ligand-binding molecule according to any one of claims 2 to 5, wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloprotease. The ligand-binding molecule according to any one of claims 2 to 5, wherein the protease cleavage sequence is a sequence including one or more sequences selected from the sequences shown in SEQ ID NOs: 2 to 82, 788 to 827, and the sequences listed in Table 1. The ligand-binding molecule according to any one of claims 1 to 7, wherein the ligand is released from the ligand-binding molecule when the cleavage site or the protease cleavage sequence is cleaved. The ligand-binding molecule according to any one of claims 1 to 8, wherein the single domain antibody is a VHH, or a single domain VH antibody, or a single domain VL antibody. The ligand-binding molecule according to any one of claims 1 to 10, wherein the ligand is a molecule having biological activity, and the single domain antibody has neutralizing activity against the ligand. The ligand binding molecule according to any one of claims 1 to 10, wherein the single domain antibody is a VHH or a single domain VH antibody, and the cleavage site or the protease cleavage sequence is introduced into one or more positions of the single domain antibody in one or more sequences selected from the following sequences:
Single domain antibody: sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering); single domain antibody: sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering); single domain antibody: sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering); single domain antibody: sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering); single domain antibody: sequence from amino acid 55 (Kabat numbering) to the sequence up to amino acid number 69 (Kabat numbering), single domain antibody, sequence from amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), single domain antibody, sequence from amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), single domain antibody, sequence from amino acid number 95 (Kabat numbering) to amino acid number 102 (Kabat numbering), single domain antibody, sequence from amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering). The ligand binding molecule according to any one of claims 1 to 10, wherein the single domain antibody is a single domain VL antibody and the cleavage site or protease cleavage sequence is introduced into one or more positions of the single domain antibody in one or more sequences selected from the following sequences:
A single domain antibody sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering), a single domain antibody sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering), a single domain antibody sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering), a single domain antibody sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering), a single domain antibody sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering), a single domain antibody sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering), a single domain antibody sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering). A complex formed of the ligand and a ligand-binding molecule according to any one of claims 1 to 12. A fusion protein in which the ligand is fused to a ligand-binding molecule according to any one of claims 1 to 12. A pharmaceutical composition comprising a ligand-binding molecule according to any one of claims 1 to 12, a complex according to claim 13, or a fusion protein according to claim 14.

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